Encapsulation of Plasmid DNA (Lipogenes) and Therapeutic Agents with Nuclear Localization Signal/Fusogenic Peptide Conjugates into Targeted Liposome Complexes

ABSTRACT

A method is disclosed for encapsulating plasmids, oligonucleotides or negatively-charged drugs into liposomes having a different lipid composition between their inner and outer membrane bilayers and able to reach primary tumors and their metastases after intravenous injection to animals and humans. The formulation method includes complex formation between DNA with cationic lipid molecules and fusogenic/NLS peptide conjugates composed of a hydrophobic chain of about 10-20 amino acids and also containing four or more histidine residues or NLS at their one end. The encapsulated molecules display therapeutic efficacy in eradicating a variety of solid human tumors including but not limited to breast carcinoma and prostate carcinoma. Combination of the plasmids, oligonucleotides or negatively-charged drugs with other anti-neoplastic drugs (the positively-charged cis-platin, doxorubicin) encapsulated into liposomes are of therapeutic value. Also of therapeutic value in cancer eradication are combinations of encapsulated the plasmids, oligonucleotides or negatively-charged drugs with HSV-tk plus encapsulated ganciclovir.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 60/210,925 filed Jun. 9, 2000. Thecontents of this application is hereby incorporated by reference intothe present disclosure.

FIELD OF THE INVENTION

The present invention relates to the field of gene therapy and isspecifically directed toward methods for producingpeptide-lipid-polynucleotide complexes suitable for delivery ofpolynucleotides to a subject. The peptide-lipid-polynucleotide complexesso produced are useful in a subject for inhibiting the progression ofneoplastic disease.

BACKGROUND OF THE INVENTION

Throughout this application various publications, patents and publishedpatent specifications are referenced by author and date or by anidentifying patent number. Full bibliographical citations for thepublications are provided immediately preceding the claims. Thedisclosures of these publications, patents and published patentspecifications are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

Gene therapy is a newly emerging field of biomedical research that holdsgreat promise for the treatment of both acute and chronic diseases andhas the potential to bring a revolutionary era to molecular medicine.However, despite numerous preclinical and clinical studies, routine useof gene therapy for the treatment of human disease has not yet beenperfected. It remains an important unmet need of gene therapy to creategene delivery systems that effectively target specific cells of interestin a subject while controlling harmful side effects.

Gene therapy is aimed at introducing therapeutically important genesinto somatic cells of patients. Diseases already shown to be amenable totherapy with gene transfer in clinical trials include, cancer (melanoma,breast, lymphoma, head and neck, ovarian, colon, prostate, brain,chronic myelogenous leukemia, non-small cell lung, lung adenocarcinoma,colorectal, neuroblastoma, glioma, glioblastoma, astrocytoma, andothers), AIDS, cystic fibrosis, adenosine deaminase deficiency,cardiovascular diseases (restenosis, familial hypercholesterolemia,peripheral artery disease), Gaucher disease, α1-antitrypsin deficiency,rheumatoid arthritis and others. Human diseases expected to be theobject of clinical trials include hemophilia A and B, Parkinson'sdisease, ocular diseases, xeroderma pigmentosum, high blood pressure,obesity. ADA deficiency was the disease successfully treated by thefirst human “gene transfer” experiment conducted by Kenneth Culver in1990. See, Culver, K. W. (1996) in: Gene Therapy: A Primer forPhysicians, Second Ed., Mary Ann Liebert, Inc. Publ, New York, pp.1-198.

The primary goals of gene therapy are to repair or replace mutatedgenes, regulate gene expression and signal transduction, manipulate theimmune system, or target malignant and other cells for destruction. See,Anderson, W. F. (1992) Science 256:808-813; Lasic, D. (1997) in:Liposomes in Gene Delivery, CRC Press, pp. 1-295; Boulikas, T. (1998)Gene Ther. Mol. Biol. 1:1-172; Martin, F. and Boulikas, T. (1998) GeneTher. Mol. Biol. 1:173-214; Ross, G. et al. (1996) Hum. Gene Ther.7:1781-1790.

Human cancer presents a particular disease condition for which effectivegene therapy methods would provide a particularly useful clinicalbenefit. Gene therapy concepts for treatment of such diseases includestimulation of immune responses as well as manipulation of a variety ofalternative cellular functions that affect the malignant phenotype.Although many human tumors are non or weakly immunogenic, the immunesystem can be reinforced and instructed to eliminate cancer cells aftertransduction of a patient's cells ex vivo with the cytokine genesGM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, and TNF-α, followed by cellvaccination of the patient (e.g. intradermally) to potentiateT-lymphocyte-mediated antitumor effects (cancer immunotherapy). DNAvaccination with genes encoding tumor antigens and immunotherapy withsynthetic tumor peptide vaccines are further developments that arecurrently being tested. The genes used for cancer gene therapy in humanclinical trials include a number of tumor suppressor genes (p53, RB,BRCA1, E1A), antisense oncogenes (antisense c-fos, c-myc, K-ras), andsuicide genes (HSV-tk, in combination with ganciclovir, cytosinedeaminase in combination with 5-fluorocytosine). Other important genesthat have been proposed for cancer gene therapy include bcl-2, MDR-1,p21, p16, bax, bcl-xs, E2F, IGF-I, VEGF, angiostatin, CFTR, LDL-R,TGF-β, and leptin. One major hurdle preventing successful implementationof these gene therapies is the difficulty of efficiently delivering aneffective dose of polynucleotides to the site of the tumor. Thus, genedelivery systems with enhanced transfection capabilities would be highlyadvantageous.

A number of different vector technologies and gene delivery methods havebeen proposed and tested for delivering genes in vivo, including viralvectors and various nucleic acid encapsulation techniques. Alternativeviral delivery vehicles for genes include murine retroviruses,recombinant adenoviral vectors, adeno-associated virus, HSV, EBV, HIVvectors, and baculovirus. Nonviral gene delivery methods use cationic orneutral liposomes, direct injection of plasmid DNA, and polymers.Various strategies to enhance efficiency of gene transfer have beentested such as fusogenic peptides in combination with liposomes orpolymers to enhance the release of plasmid DNA from endosomes.

Each of the various gene delivery techniques has been found to possessdifferent strengths and weaknesses. Recombinant retroviruses stablyintegrate into the chromosome but require host DNA synthesis to insert.Adenoviruses can infect non-dividing cells but cause immune reactionsleading to the elimination of therapeutically transduced cells.Adeno-associated virus (AAV) is not pathogenic and does not elicitimmune responses but new production strategies are required to obtainhigh AAV titers for preclinical and clinical studies. Wild-type AAVsintegrate into chromosome 19, whereas recombinant AAVs are deprived ofsite-specific integration and may also persist episomally.

Herpes Simplex Virus (HSV) vectors can infect non-replicating cells,such as neuronal cells, and has a high payload capacity for foreign DNAbut inflict cytotoxic effects. It seems that each delivery system willbe developed independently of the others and that each will demonstratestrengths and weaknesses for certain applications. At present,retroviruses are most commonly used in human clinical trials, followedby adenoviruses, cationic liposomes and AAV.

As the challenges of perfecting gene therapy techniques have becomeapparent, a variety of additional delivery systems have been proposed tocircumvent the difficulties observed with standard technologies. Forexample, cell-based gene delivery using polymer-encapsulated syngeneicor allogeneic cells implanted into a tissue of a patient can be used tosecrete therapeutic proteins. This method is being tested in trials foramyotrophic lateral sclerosis using the ciliary neurotrophic factorgene, and may be extended to Factor VIII and IX for hemophilia,interleukin genes, dopamine-secreting cells to treat Parkinson'sdisease, nerve growth factor for Alzheimer's disease and other diseases.Other techniques under development include, vectors with the Cre-LoxPrecombinase system to rid transfected cells of undesirable viral DNAsequences, use of tissue-specific promoters to express a gene in aparticular cell type, or use of ligands recognizing cell surfacemolecules to direct gene vehicles to a particular cell type.

Additional methods that have been proposed for improving the efficacy ofgene therapy technologies include designing p53 “gene bombs” thatexplode into tumor cells, exploiting the HIV-1 virus to engineer vectorsfor gene transfer, combining viruses with polymers or cationic lipids toimprove gene transfer, the attachment of nuclear localization signalpeptides to oligonucleotides to direct genes to nuclei, and thedevelopment of molecular switch systems allowing genes to be turned onor off at will. Nevertheless, because of the wide range of diseaseconditions for which gene therapies are required, and the complexitiesof developing treatments for such diseases, there remains a need forimproved techniques for performing gene therapy. The present inventionprovides methods and compositions for addressing these issues.

DISCLOSURE OF THE INVENTION

A method is disclosed for encapsulating DNA and negatively charged drugsinto liposomes having a different lipid composition between their innerand outer membrane bilayers. The liposomes are able to reach primarytumors and their metastases after intravenous injection to animals andhumans. The method includes micelle formation between DNA with a mixtureof cationic lipid and peptide molecules at molar ratios to nearlyneutralization ratios in 10-90% ethanol; the cationic peptides specifynuclear localization and have a hydrophobic moiety endowed with membranefusion to improve entrance across the cell membrane of the complex.These peptides insert with their cationic portion directed towardcondensed DNA and their hydrophobic chain buried together with thehydrophobic chains of the lipids in the micelle membrane monolayer. TheDNA/lipid/peptide micelles are converted into liposomes by mixing withpre-made liposomes or lipids followed by dilution in aqueous solutionsand dialysis to remove the ethanol and allow liposome formation andextrusion through membranes to a diameter below 160 nm entrapping andencapsulating DNA with a very high yield. The encapsulated DNA has ahigh therapeutic efficacy in eradicating a variety of solid human tumorsincluding, but not limited to, breast carcinoma and prostate carcinoma.A plasmid is constructed with DNA carrying anticancer genes including,but not limited to p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax,bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin, GM-CSF,IL-12, IL-2, IL-4, IL-7, IFN-γ, TNF-α, HSV-tk (in combination withganciclovir), E. coli cytosine deaminase (in combination with5-fluorocytosine) and is combined with encapsulated cisplatin or withother similarly systemically delivered antineoplastic drugs to suppresscancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the structure of the cancer targeted liposomecomplex.

FIG. 2 illustrates the results of plasmid DNA condensation with variousagents as well as various formulation of cationic liposomes in affectingthe level of expression of the reporter beta-galactosidase gene aftertransfection of K562 human erythroleukemia cell cultures.

FIG. 3 illustrates tumor targeting in SCID mice. FIG. 3A shows a SCIDmouse with a large and small human breast tumor before and afterstaining with X-Gal to test the expression of the transferred gene. Bothtumors turn dark blue. The intensity of the blue color is proportionalto the expression of the beta-galactosidase gene.

FIG. 3B shows that in the initial staining of the small tumor, the skinand the intestines at the injection area are the first organs to turnblue. FIG. 3C is a view of the back of the animal. The two tumors areclearly visible after removal of the skin (top). Dark staining of thesmall tumor and light blue staining of the large tumor is evident at aninitial stage of staining (bottom). FIG. 3D is a view of the front sideof the animal. The two tumors are clearly visible after removal of theskin. On the figure to the bottom the dark staining of both tumors isevident at a later stage during staining.

FIG. 3E shows the front (top) and rear (bottom) higher magnificationview of the dark staining of both tumors at a later stage duringstaining. Staining of the vascular system around the small tumor canalso be seen (bottom).

BRIEF DESCRIPTION OF THE TABLES

Table 1 is a list of molecules able to form micelles.

Table 2 lists several fusogenic peptides and describes their properties,along with a reference.

Table 3 lists simple Nuclear Localization Signal (NLS) peptides.

Table 4 shows a list of “bipartite” or “split” NLS peptides.

Table 5 lists “nonpositive NLS” peptides lacking clusters ofarginines/lysines.

Table 6 lists peptides with nucleolar localization signals (NoLS).

Table 7 lists peptides having karyophilic clusters on non-membraneprotein kinases.

Table 8 lists peptide nuclear localization signals on DNA repairproteins.

Table 9 lists NLS peptides in transcription factors.

Table 10 lists NLS peptides in other nuclear proteins.

MODES FOR CARRYING OUT THE INVENTION Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of immunology, molecular biology,microbiology, cell biology and recombinant DNA. These methods aredescribed in the following publications. See, e.g., Sambrook, et al.MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) Edition (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, F. M. Ausubel, et al. eds., (1987); theseries METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR: A PRACTICALAPPROACH, M. MacPherson, et al., IRL Press at Oxford University Press(1991); PCR 2: A PRACTICAL APPROACH, MacPherson et al., eds. (1995);ANTIBODIES, A LABORATORY MANUAL, Harlow and Lane, eds. (1988); andANIMAL CELL CULTURE, R. I. Freshney, ed. (1987).

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

The terms “polynucleotide” and “nucleic acid molecule” are usedinterchangeably to refer to polymeric forms of nucleotides of anylength. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example, single-,double-stranded and triple helical molecules, a gene or gene fragment,exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. A nucleic acid molecule may also comprise modified nucleicacid molecules.

A “gene” refers to a polynucleotide containing at least one open readingframe that is capable of encoding a particular polypeptide or proteinafter being transcribed and translated.

A “gene product” refers to the amino acid (e.g., peptide or polypeptide)generated when a gene is transcribed and translated.

The following abbreviations are used herein: DDAB: dimethyldioctadecylammonium bromide (same as N,N-distearyl-N,N-dimethylammonium bromide);DODAC: N,N-dioleyl-N,N-dimethylammonium chloride; DODAP:1,2-dioleoyl-3-dimethylammonium propane; DMRIE:N-[1′-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane;DOGS: Dioctadecylamidoglycylspermine; DOTAP (same as DOTMA):N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOSPA:N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate; DPTAP: 1,2-dipalmitoyl-3-trimethylammoniumpropane; OSTAP: 1,2-disteroyl-3-trimethylammonium propane; DOPE,1,2-sn-dioleoylphoshatidylethanolamine; DC-Chol,3β-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol. See, Gao et al.,Biochem. Biophys. Res. Comm. 179:280-285 (1991).

As used herein, the term “pharmaceutically acceptable anion” refers toanions of organic and inorganic acids that provide non-toxic salts inpharmaceutical preparations. Examples of such anions include the halidesanions, chloride, bromide, and iodide, inorganic anions such as sulfate,phosphate, and nitrate, and organic anions. Organic anions may bederived from simple organic acids, such as acetic acid, propionic acid,glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid,succinic acid, maleic, acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methane sulfonic acid,ethane sulfonic acid, p-toluenesulfonic acid, and the like. Thepreparation of pharmaceutically acceptable salts is described in Berge,et al., J. Pharm. Sci. 66:1-19 (1977), incorporated herein by reference.

Physiologically acceptable carriers, excipients or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA: sugar alcohols such as mannitol or sorbitol;salt-forming counter ions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics or polyethylene glycol (PEG). PEG moleculesalso contain a fusogenic peptide with an attached Nuclear LocalizationSignal (NLS) covalently linked to the end of the PEG molecule.

The term “cationic lipid” refers to any of a number of lipid speciesthat carry a net positive charge at physiological pH. Such lipidsinclude, but are not limited to, DDAB, DMRIE, DODAC, DOGS, DOTAP, DOSPAand DC-Chol. Additionally, a number of commercial preparations ofcationic lipids are available that can be used in the present invention.These include, for example, LIPOFECTIN (commercially available cationicliposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y.,USA); LIPOFECTAMINE (commercially available cationic liposomescomprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM(commercially available cationic lipids comprising DOGS in ethanol fromPromega Corp., Madison, Wis., USA).

This invention further provides a number of methods for producingmicelles with entrapped therapeutic drugs. The method is particularlyuseful to produce micelles of drugs or compositions having a net overallnegative charge, e.g., DNA, RNA or negatively charged small molecules.For example, the DNA can be comprised within a plasmid vector and encodefor a therapeutic protein, e.g., wild-type p53, HSV-tk, p21, Bax, Bad,IL-2, IL-12, GM-CSF, angiostatin, endostatin and oncostatin. In oneembodiment, the method requires combining an effective amount of thetherapeutic agent with an effective amount of cationic lipids. Cationiclipids useful in the methods of this invention include, but are notlimited to, DDAB, dimethyldioctadecyl ammonium bromide; DMRIE:N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammoniumbromide; DMTAP: 1,2-dimyristoyl-3-trimethylammonium propane; DOGS:Dioctadecylamidoglycylspermine; DOTAP (same as DOTMA):N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DPTAP:1,2-dipalmitoyl-3-trimethylammonium propane; DSTAP:1,2-disteroyl-3-trimethylammonium propane.

In one aspect, a ratio of from about 30 to about 90% of phosphatescontained within the negatively charged therapeutic agent areneutralized by positive charges on lipid molecules (negative charges arein excess) to form an electrostatic micelle complex in an effectiveconcentration of ethanol. In one aspect, the ethanol solution is fromabout 20% to about 80% ethanol. In a further aspect, the ethanolconcentration is about 30%. The ethanol/cationic lipid/therapeutic agentcomplex is then combined with an effective amount of afusogenic-karyophilic peptide conjugate. In one aspect, an effectiveamount of the conjugate is a ratio range from about 0.0 to about 0.3(positive charges on peptide to negative charges on phosphate groups) toneutralize the majority of the remaining negative charges on thephosphate groups of the therapeutic agents thereby leading to an almostcomplete neutralization of the complex. The optimal conditions give tothe complex a slightly negative charge. However, when the positivecharges on cationic lipids exceed the negative charges on the DNA, theexcess of positive charges are neutralized by DPPG (dipalmitoylphosphatidyl glycerol) and its derivatives, or by other anionic lipidmolecules in the final micelle complex.

In an alternative embodiment, the above methods can be modified byaddition of DNA condensing agents selected from spermine, spermidine,and magnesium or other divalent metal ions neutralizing a certainpercentage (1-20%) of phosphate groups.

In a further embodiment, the cationic lipids are combined with aneffective amount of fusogenic lipid DOPE at various molar ratios forexample, in a molar ratio of from about 1:1 cationic lipid:DOPE. In analternative embodiment, the cationic lipids are combined with aneffective amount of a fusogenic/NLS peptide conjugate. Examples offusogenic/NLS peptide conjugates include, but are not limited to(KAWLKAF)₃ (SEQ ID NO:1), GLFKAAAKLLKSLWKLLLKA (SEQ ID NO:2),LLLKAFAKLLKSLWKLLLKA (SEQ ID NO:3), as well as all derivatives of theprototype (Hydrophobic3-Karyophilic1-Hydrophobic2-Karyophilic1)₂₋₃ whereHydrophobic is any of the A, I, L, V, P, G, W, F and Karyophilic is anyof the K, R, or H, containing a positively-charged residue every 3rd or4th amino acid, which form alpha helices and direct a net positivecharge to the same direction of the helix. Additional examples includebut are not limited to GLFKAIAGFIKNGWKGMIDGGGYC (SEQ ID NO:4) frominfluenza virus hemagglutinin HA-2; YGRKKRRQRRR (SEQ ID NO:5) from TATof HIV; MSGTFGGILAGLIGLL(K/R/H)₁₋₆ (SEQ ID NO:6), derived from theN-terminal region of the S protein of duck hepatitis B virus, but withthe addition of one to six positively-charged lysine, arginine orhistidine residues, and combinations of these, able to interact directlywith the phosphate groups of plasmid or oligonucleotide DNA,compensating for part of the positive charges provided by the cationiclipids. GAAIGLAWIPYFGPAA (SEQ ID NO:7) is derived from the fusogenicpeptide of the Ebola virus transmembrane protein; residues 53-70(C-terminal helix) of apolipoprotein (apo) AII peptide; the 23-residuefusogenic N-terminal peptide of HIV-1 transmembrane glycoprotein gp41;the 29-42-residue fragment from Alzheimer's β-amyloid peptide; thefusion peptide and N-terminal heptad repeat of Sendai virus; the 56-68helical segment of lecithin cholesterol acyltransferase. Included withinthese embodiments are shorter versions of these peptides, that are knownto induce fusion of unilamellar lipid vesicles or all that are similarlyderivatized with the addition of one to six positively-charged lysine,arginine or histidine residues (K/R/H)₁₋₆ able to interact directly withthe phosphate groups of plasmid or oligonucleotide DNA, compensating forpart of the positive charges provided by the cationic lipids. Thefusogenic peptides in the fusogenic/NLS conjugates represent hydrophobicamino acid stretches, and smaller fragments of these peptide sequences,that include all signal peptide sequences used in membrane or secretedproteins that insert into the endoplasmic reticulum. Alternatively, theconjugates represent transmembrane domains and smaller fragments ofthese peptide sequences.

In one aspect of the invention, the NLS peptide component infusogenic/NLS peptide conjugates is derived from the fusogenichydrophobic peptides. However, there is an addition of 5-6 amino acidkaryophilic Nuclear Localization Signals (NLS) derived from a number ofknown NLS peptides, as well as from searches of the nuclear proteindatabases, for stretches of five or more karyophilic amino acidstretches in proteins containing at least four positively-charged aminoaids flanked by a proline (P) or glycine (G). Examples of NLS peptidesare shown in Tables 1-8. The NLS peptide component in fusogenic/NLSpeptide conjugates are synthetic peptides containing the above said NLS,but further modified by additional K, R, H residues at the central partof the peptide or with P or G at the N- or C-terminus.

In a further aspect, the fusogenic/NLS peptide conjugates are derivedfrom the said fusogenic hydrophobic peptides but with the addition of astretch of H₄₋₆ (four to six histidine residues) in the place of NLS.Micelle formation takes place at pH 5-6 where histidyl residues arepositively charged but lose their charge at the nearly neutral pH of thebiological fluids, thus releasing the plasmid or oligonucleotide DNAfrom their electrostatic interaction.

The fusogenic peptide/NLS peptide conjugates are linked to each otherwith a short amino acid stretch representing an endogenous proteasecleavage site.

In a preferred aspect of the invention, the structure of the preferredprototype fusogenic/NLS peptide conjugate used in this invention is:PKIGIRGPSP(L/A/I)₁₂₋₂₀ (SEQ ID NO:8), where (L/A/I)₁₂₋₂₀ is a stretch of12-20 hydrophobic amino acids containing A, L, I, Y, W, F and otherhydrophobic amino acids.

The micelles made by the above methods are further provided by thisinvention by conversion into liposomes. An effective amount of liposomes(diameter from about 80 to about 160 nm), or of a lipid solutioncomposed of cholesterol (from about 10% to about 50%), neutralphospholipid such as hydrogenated soy phosphatidylcholine (HSPC) (fromabout 40% to about 90%), and the derivatized vesicle-forming lipidPEG-DSPE (distearoylphosphatidyl ethanolamine) from about 1- to about 7mole percent, is added to the micelle solution.

In a specific embodiment, the liposomes are composed of vesicle-forminglipids and between from about 1 to about 7 mole percent ofdistearoylphosphatidyl ethanolamine (DSPE) derivatized with apolyethyleneglycol. The composition of claim 20, wherein thepolyethyleneglycol has a molecular weight is between about 1,000 to5,000 daltons. Micelles are converted into liposomes with a concomitantdecrease of the ethanol concentration which can be accomplished byremoval of the ethanol by dialysis of the liposome complexes throughpermeable membranes or reduced to a diameter of 80-160 nm by extrusionthrough membranes.

Liposome encapsulated therapeutic agents produced by the above methodsare further provided by this invention.

Also provided herein is a method for delivering a therapeutic agent suchas plasmid DNA or oligonucleotides to a tissue cell in vivo byintravenous, or other type of injection of the micelles or liposomes.This method specifically targets a primary tumor and the metastases bythe long circulating time of the micelle or liposome complex because ofthe exposure of PEG chains on its surface, its small size (80-160 nm)and the decrease in hydrostatic pressure in the solid tumor from thecenter to its periphery supporting a preferential extravasation throughthe tumor vasculature to the extracellular space in tumors. A method fordelivering plasmid or oligonucleotide DNA across the cell membranebarrier of the tumors using the micelle or liposome complexes describedherein is capable because of the presence of the fusogenic peptides inthe complex. In particular, a method for delivering plasmid oroligonucleotide DNA to the liver, spleen and bone marrow afterintravenous injection of the complexes is provided. Further provided isa method for delivering therapeutic genes to the liver, spleen and bonemarrow of cancer and noncancer patients including but not limited to,factor VIII or IX for the therapy of hemophilias, multidrug resistance,cytokine genes for cancer immunotherapy, genes for the alleviation ofpain, genes for the alleviation of diabetes and genes that can beintroduced to liver, spleen and bone marrow tissue, to produce asecreted form of a therapeutic protein.

The disclosed therapies also provide methods for reducing tumor size bycombining the encapsulated plasmid DNA carrying one or more anticancergenes selected from the group consisting of p53, RB, BRCA1, E1A, bcl-2,MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin,endostatin, GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-γ, TNF-α, HSV-tk (incombination with ganciclovir), E. coli cytosine deaminase (incombination with 5-fluorocytosine) with encapsulated antisenseoligonucleotides (antisense c-fos, c-myc, K-ras), ribozymes ortriplex-forming oligonucleotides directed against genes that control thecell cycle or signaling pathways. These methods can be modified bycombining the encapsulated plasmid DNA carrying one or more anticancergenes of with encapsulated or free antineoplastic drugs, consisting ofthe group of adriamycin, angiostatin, azathioprine, bleomycin,busulfane, camptothecin, carboplatin, carmustine, chlorambucile,chlormethamine, chloroquinoxaline sulfonamide, cisplatin,cyclophosphamide, cycloplatam, cytarabine, dacarbazine, dactinomycin,daunorubicin, didox, doxorubicin, endostatin, enloplatin, estramustine,etoposide, extramustinephosphat, flucytosine, fluorodeoxyuridine,fluorouracil, gallium nitrate, hydroxyurea, idoxuridine, interferons,interleukins, leuprolide, lobaplatin, lomustine, mannomustine,mechlorethamine, mechlorethaminoxide, melphalan, mercaptopurine,methotrexate, mithramycin, mitobronitole, mitomycin, mycophenolic acid,nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine,platinum-triamine complex, plicamycin, prednisolone, prednisone,procarbazine, protein kinase C inhibitors, puromycine, semustine, signaltransduction inhibitors, spiroplatin, streptozotocine, stromelysininhibitors, taxol, tegafur, telomerase inhibitors, teniposide,thalidomide, thiamiprine, thioguanine, thiotepa, tiamiprine, tretamine,triaziquone, trifosfamide, tyrosine kinase inhibitors, uramustine,vidarabine, vinblastine, vinca alcaloids, vincristine, vindesine,vorozole, zeniplatin, zeniplatin, and zinostatin.

The following examples are intended to illustrate, but not limit theinvention.

Liposome Composition

Liposomes are microscopic vesicles consisting of concentric lipidbilayers. Structurally, liposomes range in size and shape from longtubes to spheres, with dimensions from a few hundred Angstroms tofractions of a millimeter. Vesicle-forming lipids are selected toachieve a specified degree of fluidity or rigidity of the final complexproviding the lipid composition of the outer layer. These are neutral(cholesterol) or bipolar and include phospholipids, such asphosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI), and sphingomyelin (SM) and other type ofbipolar lipids including but not limited todioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain lengthin the range of 14-22, and saturated or with one or more double C═Cbonds. Examples of lipids capable of producing a stable liposome, alone,or in combination with other lipid components are phospholipids, such ashydrogenated soy phosphatidylcholine (HSPC), lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebro sides,distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE) anddioleoylphosphatidylethanolamine4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal). Additionalnon-phosphorous containing lipids that can become incorporated intoliposomes include stearylamine, dodecylamine, hexadecylamine, isopropylmyristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetylpalmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylicpolymers, polyethyloxylated fatty acid amides, and the cationic lipidsmentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA,DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidicacid (PA), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are ableto form vesicles. Preferred lipids for use in the present invention arecholesterol, hydrogenated soy phosphatidylcholine (HSPC) and, thederivatized vesicle-forming lipid PEG-DSPE.

Typically, liposomes can be divided into three categories based on theiroverall size and the nature of the lamellar structure. The threeclassifications, as developed by the New York Academy Sciences Meeting,“Liposomes and Their Use in Biology and Medicine,” December 1977, aremulti-lamellar vesicles (MLVs), small uni-lamellar vesicles (SUVs) andlarge uni-lamellar vesicles (LUVs).

SUVs range in diameter from approximately 20 to 50 nm and consist of asingle lipid bilayer surrounding an aqueous compartment. Unilamellarvesicles can also be prepared in sizes from about 50 nm to 600 nm indiameter. While unilamellar are single compartmental vesicles of fairlyuniform size, MLVs vary greatly in size up to 10,000 nm, or thereabouts,are multi-compartmental in their structure and contain more than onebilayer. LUV liposomes are so named because of their large diameter thatranges from about 600 nm to 30,000 nm; they can contain more than onebilayer.

Liposomes may be prepared by a number of methods not all of whichproduce the three different types of liposomes. For example, ultrasonicdispersion by means of immersing a metal probe directly into asuspension of MLVs is a common way for preparing SUVs.

Preparing liposomes of the MLV class usually involves dissolving thelipids in an appropriate organic solvent and then removing the solventunder a gas or air stream. This leaves behind a thin film of dry lipidon the surface of the container. An aqueous solution is then introducedinto the container with shaking, in order to free lipid material fromthe sides of the container. This process disperses the lipid, causing itto form into lipid aggregates or liposomes. Liposomes of the LUV varietymay be made by slow hydration of a thin layer of lipid with distilledwater or an aqueous solution of some sort. Alternatively, liposomes maybe prepared by lyophilization. This process comprises drying a solutionof lipids to a film under a stream of nitrogen. This film is thendissolved in a volatile solvent, frozen, and placed on a lyophilizationapparatus to remove the solvent. To prepare a pharmaceutical formulationcontaining a drug, a solution of the drug is added to the lyophilizedlipids, whereupon liposomes are formed.

Preparing Cationic Liposome/Cationic Peptide/Nucleic Acid Micelles

Cationic lipids, with the exception of sphingosine and some lipids inprimitive life forms, do not occur in nature. The present invention usessingle-chain amphiphiles which are chloride and bromide salts of thealkyltrimethylammonium surfactants including but not limited to C12 andC16 chains abbreviated DDAB (same as DODAB) or CTAB. The moleculargeometry of these molecules determines the critical micelleconcentration (ratio between free monomers in solution and molecules inmicelles). Lipid exchange between the two states is a highly dynamicprocess; phospholipids have critical micelle concentration values below10⁻⁸ M and are more stable in liposomes; however, single chaindetergents, such as stearylamine, may emerge from the liposome membraneupon dilution or intravenous injection in milliseconds (Lasic, 1997).

Cationic lipids include, but are not limited to, DDAB:dimethyldioctadecyl ammonium bromide (same asN,N-distearyl-N,N-dimethylammonium bromide); DMRIE:N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammoniumbromide; DODAC: N,N-dioleyl-N,N-dimethylammonium chloride; DMTAP:1,2-dimyristoyl-3-trimethylammonium propane; DODAP:1,2-dioleoyl-3-dimethylammonium propane; DOGS:Dioctadecylamidoglycylspermine; DOTAP (same as DOTMA):N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride; DOSPA:N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate; DPTAP: 1,2-dipalmitoyl-3-trimethylammoniumpropane; DSTAP: 1,2-disteroyl-3-trimethylammonium propane; DC-Chol,3β-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol.

Lipid-based vectors used in gene transfer have been formulated in one oftwo ways. In one method, the nucleic acid is introduced into preformedliposomes made of mixtures of cationic lipids and neutral lipids. Thecomplexes thus formed have undefined and complicated structures and thetransfection efficiency is severely reduced by the presence of serum.Preformed liposomes are commercially available as LIPOFECTIN andLIPOFECTAMINE. The second method involves the formation of DNA complexeswith mono- or poly-cationic lipids without the presence of a neutrallipid. These complexes are prepared in the presence of ethanol and arenot stable in water. Additionally, these complexes are adverselyaffected by serum (see, Behr, Acc. Chem. Res. 26:274-78 (1993)). Anexample of a commercially available poly-cationic lipid is TRANSFECTAM.Other efforts to encapsulate DNA in lipid-based formulations have notovercome these problems (see, Szoka et al., Ann. Rev. Biophys. Bioeng.9:467 (1980); and Deamer, U.S. Pat. No. 4,515,736).

The nucleotide polymers can be single-stranded DNA or RNA, ordouble-stranded DNA or DNA-RNA hybrids. Examples of double-stranded DNAinclude structural genes, genes including control and terminationregions, and self-replicating systems such as plasmid DNA. Particularlypreferred nucleic acids are plasmids. Single-stranded nucleic acidsinclude antisense oligonucleotides (complementary to DNA and RNA),ribozymes and triplex-forming oligonucleotides. In order to increasestability, some single-stranded nucleic acids will preferably have someor all of the nucleotide linkages substituted with stable,non-phosphodiester linkages, including, for example, phosphorothioate,phosphorodithioate, phosphoroselenate, methylphosphonate, or O-alkylphosphotriester linkages.

Encapsulating Cationic Liposome/Cationic Peptide/Nucleic Acid Micellesinto Neutral Liposomes

Cationic lipids used with fusogenic peptide/NLS conjugates to providethe inner layer of the particle can be any of a number of substancesselected from the group of DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP(DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol. The cationic lipid is combinedwith DOPE. In one group of embodiments, the preferred cationic lipid isDDAB:DOPE 1:1.

Neutral lipids used herein to provide the outer layer of the particlescan be any of a number of lipid species that exist either in anuncharged or neutral zwitterionic form at physiological pH. Such lipidsare selected from a group consisting of diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, andcerebrosides. In one group of embodiments, lipids containing saturated,mono-, or di-unsaturated fatty acids with carbon chain lengths in therange of C14 to C22 are preferred. In general, less saturated lipids aremore easily sized, particularly when the liposomes must be sized belowabout 0.16 microns, for purposes of filter sterilization. Considerationof liposome size, rigidity and stability of the liposomes in the finalpreparation, its shelf life without leakage of the encapsulated DNA, andstability in the bloodstream generally guide the selection of neutrallipids for providing the outer coating of our gene vehicles. Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In another group of embodiments, lipids withcarbon chain lengths in the range of C14 to C22 are used. Preferably,the neutral lipids used in the present invention are hydrogenated soyphosphatidylcholine (HSPC), cholesterol, and PEG-distearoylphosphatidylethanolamine (DSPE) or PEG-ceramide.

Methods for Preparing Liposomes

A variety of methods for preparing various liposome forms have beendescribed in several issued patents, for example, U.S. Pat. Nos.4,229,360; 4,224,179; 4,241,046; 4,737,323; 4,078,052; 4,235,871;4,501,728; and 4,837,028, as well as in the articles Szoka et al., Ann.Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al., Chem. Phys. Lip.40:89 (1986). These methods do not produce all three different types ofliposomes (MLVs, SUVs, LUVs). For example, ultrasonic dispersion bymeans of immersing a metal probe directly into a suspension of MLVs is acommon way for preparing SUVs.

Preparing liposomes of the MLV class usually involves dissolving thelipids in an appropriate organic solvent and then removing the solventunder a gas or air stream. This leaves behind a thin film of dry lipidon the surface of the container. An aqueous solution is then introducedinto the container with shaking, in order to free lipid material fromthe sides of the container. This process disperses the lipid, causing itto form into lipid aggregates or liposomes. Liposomes of the LUV varietymay be made by slow hydration of a thin layer of lipid with distilledwater or an aqueous solution of some sort. Alternatively, liposomes maybe prepared by lyophilization. This process comprises drying a solutionof lipids to a film under a stream of nitrogen. The film is thendissolved in a volatile solvent, frozen, and placed on a lyophilizationapparatus to remove the solvent. To prepare a pharmaceutical formulationcontaining a drug, a solution of the drug is added to the lyophilizedlipids, whereupon liposomes are formed.

Following liposome preparation, the liposomes may be sized to achieve adesired size range and relatively narrow distribution of liposome sizes.Preferably, the preformed liposomes are sized to a mean diameter ofabout 80 to 160 nm (the upper size limit for filter sterilization beforein vivo administration). Several techniques are available for sizingliposomes to a desired size. Sonicating a liposome suspension either bybath or probe sonication produces a progressive size reduction down tosmall unilamellar vesicles less than about 0.05 microns (50 nm) in size.Extrusion of liposome through a small-pore polycarbonate is ourpreferred method for reducing liposome sizes to a relativelywell-defined size distribution. The liposomes may be extruded throughsuccessively smaller-pore membranes, to achieve a gradual reduction inliposome size.

One way used to coat DNA with lipid is by controlled detergent depletionfrom a cationic lipid/DNA/detergent complex. This method can givecomplexes with stability in plasma. Hofland et al. (1996), have preparedsuch complexes by dialysis of a mixture ofDOSPA/DOPE/DNA/octylglucoside.

Pharmaceutical compositions comprising the cationic liposome/nucleicacid complexes of the invention are prepared according to standardtechniques and further comprise a pharmaceutically acceptable carrier.Generally, normal saline will be employed as the pharmaceuticallyacceptable carrier.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intravenously,intraperitoneally, subcutaneously, intrathecally, injection to thespinal cord, intramuscularly, intraarticularly, portal vein injection,or intratumorally. More preferably, the pharmaceutical compositions areadministered intravenously or intratumorally by a bolus injection. Inother methods, the pharmaceutical preparations may be contacted with thetarget tissue by direct application of the preparation to the tissue.The application may be made by topical “open” or “closed” procedures.The term “topical” means the direct application of the pharmaceuticalpreparation to a tissue exposed to the environment, such as the skin, toany surface of the body, nasopharynx, external auditory canal, ocularadministration and administration to the surface of any body cavities,inhalation to the lung, genital mucosa and the like.

“Open” procedures are those procedures that include incising the skin ofa patient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue.

“Closed” procedures are invasive procedures in which the internal targettissues are not directly visualized, but accessed via insertion ofinstruments through small wounds in the skin. For example, thepreparations may be administered to the peritoneum by needle lavage.Likewise, the pharmaceutical preparations may be administered to themeninges or spinal cord by infusion during a lumbar puncture followed byappropriate positioning of the patient as commonly practiced for spinalanesthesia or metrazamide imaging of the spinal cord. Alternatively, thepreparations may be administered through endoscopic devices.

EXAMPLES Materials and Methods

DDAB, DOPE (dioleoylphosphatidylethanolamine) and most other lipids usedhere were purchased from Avanti Polar Lipids; PEG-DSPE was from Syngena.

Engineering of Plasmid pLF

The pGL3-C (Promega) was cut with XbaI and blunt-end ligated using theKlenow fragment of E. coli DNA polymerase. It was then cut with HindIIIand the 1689-bp fragment, carrying the luciferase gene, wasgel-purified. The pGFP-N1 plasmid (Clontech) was cut with SmaI andHindIII and the 4.7 kb fragment, isolated from an agarose gel, wasligated with the luciferase fragment. JM109 E. coli cells weretransformed and 20 colonies were selected; about half of them showed thepresence of inserts; 8 clones with inserts were cut with BamHI and XhoIto further confirm the presence of the luciferase gene; seven of themwere positive.

Radiolabeled plasmid pLF was generated by culturing Escherichia coli in³H-thymidine-5′-triphosphate or ³²P inorganic phosphate (5 mCi)(Dupont/NEN, Boston, Mass.) and purified using standard techniques asdescribed above.

DLS Measurements

A Coulter N4M light scattering instrument was used, at a 90° angle, setat a run time of 200 sec, using 4 to 25 microsec sample time. The scanof the particle size distribution was obtained in 1 ml sample volumeusing plastic cuvettes, at 20° C. and at 0.01 poise viscosity.

In one aspect, this invention provides a method for entrapping DNA intolipids that enhances the content of plasmid per volume unit, and reducesthe toxicity of the cationic lipids used to trap plasmid oroligonucleotide DNA. The DNA becomes hidden in the inner membranebilayer of the final complex. Furthermore, the gene transfer complex isendowed with long circulation time in body fluids and extravasatespreferentially into solid tumors and their metastatic foci and nodules.The extravasation occurs through their vasculature at most sites of thehuman or animal body after intravenous injection of the gene-carryingvehicles. This occurs because of their small size (100-160 nm), theircontent in neutral to slightly negatively-charged lipids in their outermembrane bilayers, and their coating with PEG. These gene deliveryvehicles are able to cross the cell membrane barrier after they reachthe extracellular tumor space because of the presence of fusogenicpeptides conjugated with karyophilic peptides. The vehicles assume acertain predefined orientation in the lipid membrane with their positiveends directed toward DNA and their hydrophobic tail buried inside thehydrophobic lipid bilayer. The labile NLS-fusogenic peptide linkage iscleaved after endocytosis and the remaining NLS peptide bound to plasmidDNA aids its nuclear uptake. This occurs especially when non-dividingcells are targeted, such as liver, spleen or bone marrow cells thatrepresent the major sites for extravasation and concentration of thesevehicles other than solid tumors.

Organic Solvent

A suitable solvent for preparing a micelle from the desired lipidcomponents is ethanol, methanol, or other aliphatic alcohols such aspropanol, isopropanol, butanol, tert-butanol, iso-butanol, pentanol andhexanol. Mixtures of two or more solvents may be used in the practice ofthe invention. It is also to be understood that any solvent that ismiscible with an ethanol solution, even in small amounts, can be used toimprove micelle formation and its subsequent conversion into liposomes,including chloroform, dichloromethane, di ethylether, cyclohexane,cyclopentane, benzene, and toluene.

Cationic Lipids

In a further embodiment, the liposome encapsulated DNA described hereinfurther comprises an effective amount of cationic lipids. Cationiclipids have been widely used for gene transfer; a number of clinicaltrials (34 out of 220 total RAC-approved protocols as of December, 1997)use cationic lipids. Although many cell culture studies have beendocumented, systemic delivery of genes with cationic lipids in vivo hasbeen very limited. All clinical protocols use subcutaneous, intradermal,intratumoral, and intracranial injection as well as intranasal,intrapleural, or aerosol administration but not I.V. delivery, becauseof the toxicity of the cationic lipids and DOPE (see, Martin andBoulikas, 1998). Liposomes formulated from DOPE and cationic lipidsbased on diacyltrimethylammonium propane (dioleoyl-, dimyristoyl-,dipalmitoyl-, disteroyl-trimethylammonium propane or DOTAP, DMTAP,DPTAP, DSTAP, respectively) or DDAB were highly toxic when incubated invitro with phagocytic cells (macrophages and U937 cells), but nottowards non-phagocytic T lymphocytes. The rank order of toxicity wasDOPE/DDAB>DOPE/DOTAP>DOPE/DMTAP>DOPE/DPTAP>DOPE/DSTAP; and the toxicitywas determined from the effect of the cationic liposomes on thesynthesis of nitric oxide (NO) and TNF-α produced by activatedmacrophages (Filion and Phillips, 1997).

Another aspect to be considered before I.V. injection is undertaken, isthat negatively charged serum proteins can interact and causeinactivation of cationic liposomes (Yang and Huang, 1997). Condensingagents used for plasmid delivery including polylysine,transferrin-polylysine, a fifth-generation poly(amidoamine) (PAMAM)dendrimer, poly(ethyleneimine), and several cationic lipids (DOTAP,DC-Chol/DOPE, DOGS/DOPE, and DOTMA/DOPE), were found to activate thecomplement system to varying extents. Strong complement activation wasseen with long-chain polylysines, the dendrimer, poly(ethyleneimine),and DOGS. Modifying the surface of preformed DNA complexes withpolyethyleneglycol (Plank et al., 1996) considerably reduced complementactivation.

Cationic lipids increase the transfection efficiency by destabilizingthe biological membranes, including plasma, endosomal, and lysosomalmembranes. Incubation of isolated lysosomes with low concentrations ofDOTAP caused a striking increase in free activity of β-galactosidase,and even a release of the enzyme into the medium. This demonstrates thatthe lysosomal membrane is deeply destabilized by the lipid. Themechanism of destabilization was thought to involve an interactionbetween cationic liposomes and anionic lipids of the lysosomal membrane,thus allowing a fusion between the lipid bilayers. The process was lesspronounced at pH 5 than at pH 7.4, and anionic amphipathic lipids wereable to prevent partially this membrane destabilization (Wattiaux etal., 1997).

In contrast to DOTAP and DMRIE that were 100% charged at pH 7.4, DC-CHOLwas only about 50% charged as monitored by a pH-sensitive fluorophore.

This difference decreases the charge on the external surfaces of theliposomes, and was proposed to promote an easier dissociation ofbilayers containing DC-CHOL from the plasmid DNA, and an increase inrelease of the DNA-lipid complex into the cytosol from the endosomes(Zuidam and Barenholz, 1997).

Although cationic lipids have been used widely for the delivery ofgenes, very few studies have used systemic I.V. injection of cationicliposome-plasmid complexes. This is because of the toxicity of the lipidcomponent in animal models, not humans. Administration by I.V. injectionof two types of cationic lipids of similar structure, DOTMA and DOTAP,shows that the transfection efficiency is determined mainly by thestructure of the cationic lipid and the ratio of cationic lipid to DNA;the luciferase and GFP gene expression in different organs wastransient, with a peak level between 4 and 24 hr, dropping to less than1% of the peak level by day 4 (Song et al., 1997).

A number of different organs in vivo can be targeted after liposomaldelivery of genes or oligonucleotides. Intravenous injection of cationicliposome-plasmid complexes by tail vein in mice, targeted mainly thelung and to a smaller extent the liver, spleen, heart, kidney and otherorgans (Zhu et al., 1993). Intraperitoneal injection of aplasmid-liposome complex expressing antisense K-ras RNA in nude miceinoculated i.p. with AsPC-1 pancreatic cancer cells harboring K-raspoint mutations and PCR analysis indicated that the injected DNA wasdelivered to various organs except brain (Aoki et al., 1995).

A number of factors for DOTAP:cholesterol/DNA complex preparationincluding the DNA:liposome ratio, mild sonication, heating, andextrusion were found to be crucial for improved systemic delivery;maximal gene expression was obtained when a homogeneous population ofDNA:liposome complexes between 200 to 450 nm in size were used.Cryo-electron microscopy showed that the DNA was condensed on theinterior of invaginated liposomes between two lipid bilayers in theseformulations, a factor that was thought to be responsible for the hightransfection efficiency in vivo and for the broad tissue distribution(Templeton et al., 1997).

Steps to improve liposome-mediated gene delivery to somatic cellsinclude, persistence of the plasmid in blood circulation, port of entryand transport across the cell membrane, release from endosomalcompartments into the cytoplasm, nuclear import by docking through thepore complexes of the nuclear envelope, expression driven by theappropriate promoter/enhancer control elements, and persistence of theplasmid in the nucleus for long periods (Boulikas, 1998a).

Plasmid Condensation with Spermine

In a further embodiment, the liposome encapsulated DNA described hereinis condensed with spermine and/or spermidine. DNA can be presented tocells in culture as a complex with polycations such as polylysine, orbasic proteins such as protamine, total histones or specific histonefractions, protamine (Boulikas and Martin, 1997). The interaction ofplasmid DNA with protamine sulfate, followed by the addition of DOTAPcationic liposomes, offered a better protection of plasmid DNA againstenzymatic digestion. The method gave consistently higher gene expressionin mice via tail vein injection as compared with DOTAP/DNA complexes. 50μg of luciferase-plasmid per mouse gave 20 ng luciferase protein per mgextracted tissue protein in the lung, that was detected as early as 1 hafter injection, peaked at 6 h and declined thereafter. Intraportalinjection of protamine/DOTAP/DNA led to about a 100-fold decrease ingene expression in the lung as compared with I.V. injection. Endothelialcells were the primary locus of lacZ transgene expression (Li and Huang,1997). Protamine sulfate enhanced plasmid delivery into severaldifferent types of cells in vitro, using the monovalent cationicliposomal formulations (DC-Chol and lipofectin). This effect was lesspronounced with the multivalent cationic liposome formulation,lipofectamine (Sorgi et al., 1997).

Spermine is found to enhance the transfection efficiency of DNA-cationicliposome complexes in cell culture and in animal studies. This biogenicpolyamine at high concentrations caused liposome fusion most likelypromoted by the simultaneous interaction of one molecule of spermine(four positively charged amino groups) with the polar head groups of twoor more molecules of lipids. At low concentrations (0.03-0.1 mM) itpromoted anchorage of the liposome-DNA complex to the surface of cellsand enhanced significantly transfection efficiency (Boulikas,unpublished).

The polycations polybrene, protamine, DEAE-dextran, and poly-L-lysinesignificantly increased the efficiency of adenovirus-mediated genetransfer in cell culture. This was thought to act by neutralizing thenegative charges presented by membrane glycoproteins that reduce theefficiency of adenovirus-mediated gene transfer (Arcasoy et al., 1997).

Oligonucleotide Transfer

In a further embodiment, the liposome encapsulates oligonucleotide DNA.Encapsulation of oligonucleotides into liposomes increased theirtherapeutic index, prevented degradation in cultured cells, and in humanserum and reduced toxicity to cells (Thierry and Dritschilo, 1992;Capaccioli et al., 1993; Lewis et al., 1996). However, most studies havebeen performed in cell culture, and very few in animals in vivo. Thereare still an important number of improvements needed before theseapproaches can move into clinical studies.

Zelphati and Szoka (1997), have found that complexes of fluorescentlylabeled oligonucleotides with DOTAP liposomes, entered the cell using anendocytic pathway mainly involving uncoated vesicles. Oligonucleotideswere redistributed from punctate cytoplasmic regions into the nucleus.This process was independent of acidification of the endosomal vesicles.The nuclear uptake of oligonucleotides depended on several factors, suchas charge of the particle, where positively charged complexes wererequired for enhanced nuclear uptake. DOTAP increased over 100 fold theantisense activity of a specific anti-luciferase oligonucleotide.Physicochemical studies of oligonucleotide-liposome complexes ofdifferent cationic lipid compositions indicated that eitherphosphatidylethanolamine or negative charges on other lipids in the cellmembrane are required for efficient fusion with cationicliposome-oligonucleotide complexes to promote entry to the cell(Jaaskelainen et al., 1994).

Similar results were reported by Lappalainen et al. (1997).Digoxigenin-labeled oligodeoxynucleotides (ODNs) complexed with thepolycationic DOSPA and the monocationic DDAB (with DOPE as a helperlipid) were taken up by CaSki cells in culture by endocytosis. Thenuclear membrane was found to pose a barrier against nuclear import ofODNs that accumulated in the perinuclear area. Although DOSPA/DOPEliposomes could deliver ODNs into the cytosol, they were unable tomediate nuclear import of ODNs. On the contrary,oligonucleotide-DDAB/DOPE complexes with a net positive charge werereleased from vesicles into the cytoplasm. It was determined thatDDAB/DOPE mediated nuclear import of the oligonucleotides.

DOPE-heme (ferric protoporphyrin IX) conjugates, inserted in cationiclipid particles with DOTAP, protected oligoribonucleotides fromdegradation in human serum and increased oligoribonucleotide uptake into2.2.15 human hepatoma cells. The enhancing effect of heme was evidentonly at a net negative charge in the particles (Takle et al., 1997).Uptake of liposomes labeled with ¹¹¹In and composed of DC-Chol and DOPEwas primarily by liver, with some accumulation in spleen and skin andvery little in the lung after I.V. tail injection. Preincubation ofcationic liposomes with phosphorothioate oligonucleotide induced adramatic, yet transient, accumulation of the lipid in lung thatgradually redistributed to liver. The mechanism of lung uptake involvedentrapment of large aggregates of oligonucleotides within pulmonarycapillaries at 15 min post-injection via embolism. Labeledoligonucleotide was localized primarily to phagocytic vacuoles ofKupffer cells at 24 h post-injection. Nuclear uptake of oligonucleotidesin vivo was not observed (Litzinger et al., 1996).

Polyethylene Glycol (PEG)-Coated Liposomes

In a further embodiment, the liposome encapsulated DNA described herein,further comprise coating of the final complex in step 2 (FIG. 1) withPEG. It is often desirable to conjugate a lipid to a polymer thatconfers extended half-life, such as polyethylene glycol (PEG).Derivatized lipids that are employed, include PEG-modified DSPE orPEG-ceramide. Addition of PEG components prevents complex aggregation,increases circulation lifetime of particles (liposomes, proteins, othercomplexes, drugs) and increases the delivery of lipid-nucleic acidcomplexes to the target tissues. See, Maxfield et al., Polymer16:505-509 (1975); Bailey, F. E. et al., in: Nonionic Surfactants,Schick, M. J., ed., pp. 794-821 (1967); Abuchowski, A. et al., J. Biol.Chem. 252:3582-3586 (1977); Abuchowski, A. et al., Cancer Biochem.Biophys. 7:175-186 (1984); Katre, N. V. et al., Proc. Natl. Acad. Sci.USA 84:1487-1491 (1987); Goodson, R. et al. Bio Technology 8:343-346(1990).

Conjugation to PEG is reported to have reduced immunogenicity andtoxicity. See, Abuchowski et al., J. Biol. Chem. 252:3578-3581 (1977).The extent of enhancement of blood circulation time of liposomes, bycoating with PEG is described in U.S. Pat. No. 5,013,556. Typically, theconcentration of the PEG-modified phospholipids, or PEG-ceramide in thecomplex will be about 1-7%. In a particularly preferred embodiment, thePEG-modified lipid is a PEG-DSPE.

Coating the surface of liposomes with inert materials designed tocamouflage the liposome from the body's host defense systems was shownto increase remarkably the plasma longevity of liposomes. The biologicalparadigm for this “surface modified” sub-branch was the erythrocyte, acell that is coated with a dense layer of carbohydrate groups, and thatmanages to evade immune system detection and to circulate for severalmonths (before being removed by the same type of cell responsible forremoving liposomes).

The first breakthrough came in 1987 when a glycolipid (the braintissue-derived ganglioside GM1), was identified that, when incorporatedwithin the lipid matrix, allowed liposomes to circulate for many hoursin the blood stream (Allen and Chonn, 1987). A second glycolipid,phosphatidylinositol, was also found to impart long plasma residencetimes to liposomes and, since it was extracted from soybeans, not braintissue, was believed to be a more pharmaceutically acceptable excipient(Gabizon et al., 1989).

A major advance in the surface-modified sub-branch was the developmentof polymer-coated liposomes (Allen et al. 1991). Polyethylene glycol(PEG) modification had been used for many years to prolong thehalf-lives of biological proteins (such as enzymes and growth factors)and to reduce their immunogenicity (e.g. Beauchamp et al., 1983). It wasreported in the early 1990s that PEG-coated liposomes circulated forremarkably long times after intravenous administration. Half-lives onthe order of 24 h were seen in mice and rats, and over 30 hours in dogs.The term “stealth” was applied to these liposomes because of theirability of evade interception by the immune system. The PEG hydrophilicpolymers form dense “conformational clouds” to prevent othermacromolecules from interaction with the surface, even at lowconcentrations of the protecting polymer (Gabizon and Papahadjopoulos,1988; Papahadjopoulos et al., 1991; reviewed by Torchilin, 1998). Theincreased hydrophilicity of the liposomes after their coating with theamphipathic PEG5000 leads to a reduction in nonspecific uptake by thereticuloendothelial system.

Whereas the half-life of antimyosin immunoliposomes was 40 min, bycoating with PEG, they increased their half-life to 1000 min afterintravenous injection to rabbits (Torchilin et al., 1992).

Micelles, Surfactants and Small Unilamellar Vesicles

In a further embodiment, the liposome encapsulated DNA described herein,further comprise an initial step of micelle formation between cationiclipids and condensed plasmid or oligonucleotide DNA in ethanolsolutions. Micelles are small amphiphilic colloidal particles formed bycertain kinds of lipid molecules, detergents or surfactants underdefined conditions of concentration, solvent and temperature. They arecomposed of a single lipid layer. Micelles can have their hydrophilichead groups assembled exposing their hydrophobic tails to the solvent(for example in 30-60% aqueous ethanol solution) or can reverse theirstructures exposing their polar heads toward the solvent such as bylowering the concentration of the ethanol to below 10% (reversemicelles). Micelle systems are in thermodynamic equilibrium with thesolvent molecules and environment. This results in constant phasechanges, especially upon contact with biological materials, such as uponintroduction to cell culture, injection to animals, dilution, contactwith proteins or other macromolecules. These changes result in rapidmicelle disassembly or flocculation. This is in contrast to the muchhigher stability of liposome bilayers.

Single-chain surfactants are able to form micelles (see Table 1, below).These include the anionic (sodium dodecyl sulfate, cholate or oleate) orcationic (cetyl-trimethylammonium bromide, CTAB) surfactants. CTAB,CTAC, and DOIC micelles yielded larger solubility gaps (lowerconcentration of colloidally suspended DNA) than corresponding SUVparticles containing neutral lipid and CTAB (1:1) (Lasic, 1997).

TABLE 1 Molecules able to form micelles Molecule Reference CTAB, CTAC,DOIC Lasic, 1997 Detergent/phospholipid micelles Lusa et al., 1998Dodecyl betaine de la Maza et al., 1998 (amphoteric surfactant)Dodecylphosphocholine cholate Lasic, 1997 Glycine-conjugated bile saltLeonard and Cohen, 1998 (anionic steroid detergent-like molecule)Lipid-dodecyl maltoside micelles Lambert et al., 1998 mixed micellesLopez et al., 1998 (Triton X-100 & phosphatidylcholine) OctylglucosideLeonard and Cohen, 1998 (non-ionic straight chain detergent) OleateLasic, 1997 PEG- dialkylphosphatidic acid Tirosh et al., 1998(dihexadecylphosphatidyl (DHP)- PEG2000) Phosphatidylcholine Schroederet al., 1990 (neutral zwitterionic) Polyethyleneglycol (MW 5000)-Weissig et al., 1998 distearoyl phosphatidyl ethanolamine (PEG-DSPE)sodium dodecyl sulfate Leonard and Cohen, 1998 (anionic straight chaindetergent) Sodium taurofusidate Leonard and Cohen, 1998 (conjugatedfungal bile salt analog) Taurine- conjugated bile salts Leonard andCohen, 1998 (anionic steroid detergent-like molecule) Triton X-100surfactant Lasic, 1997

There is a critical detergent/phospholipid ratio at whichlamellar-to-micellar transition occurs. For example, the vesicle-micelletransition was observed for dodecyl maltoside with large unilamellarliposomes. A striking feature of the solubilization process by dodecylmaltoside was the discovery of a new phase, consisting of a very viscous“gel-like” structure composed of long filamentous thread-like micelles,over 1 to 2 microns in length.

A long circulating complex needs to be slightly anionic. Therefore theliposomes used for the conversion of the micelles into liposomes containbipolar lipids (PC, PE) and 1-30% negatively charged lipids (DPPG). Thecationic lipids which are toxic, are hidden in the inner liposomemembrane bilayer. Those reaching the solid tumor will exert their toxiceffects causing apoptosis. Apoptosis will be caused by the delivery ofthe toxic drug or anti-neoplastic gene or oligonucleotide to the cancercell but also by the nuclear localization of the cationic lipids (alongwith plasmid DNA) to the nucleus. Indeed, a number of studies suggestthat plasmid DNA is imported to nuclei; its translocation docks cationiclipid molecules electrostatically attached to the DNA. These cationiclipid molecules exert their toxicity by interfering with the nucleosomeand domain structure of the chromatin causing local destabilization.This disturbance or aberrant chromatin reorganization could be exertedat the level of the nuclear matrix where plasmid DNA is attached fortranscription, autonomous replication, or integration via recombination.

Surfactants have found wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The use of surfactants in drugproducts, formulations and in emulsions has been reviewed (Rieger, in:Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, 1988, p.285).

Nonionic surfactants find wide application in pharmaceutical andcosmetic products and are usable over a wide range of pH values. Ingeneral, their HLB values range from 2 to about 18, depending on theirstructure. Nonionic surfactants include, nonionic esters such asethylene glycol esters, propylene glycol esters, glyceryl esters,polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylatedesters. Nonionic alkanolamides and ethers, such as fatty alcoholethoxylates, propoxylated alcohols, and ethoxylated/propoxylated, blockpolymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

Anionic surfactants include carboxylates such as soaps, acyl lactylates,acyl amides of amino acids, esters of sulfuric acid such as alkylsulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

Cationic surfactants include quaternary ammonium salts and ethoxylatedamines. The quaternary ammonium salts are the most used members of thisclass. If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

Classical micelles may not be effective as gene transfer vehicles, butimportant intermediates in the formation of liposome complexesencapsulating drugs or nucleic acids. The stability of single chainsurfactants-DNA-colloidal systems is lower than SUV particles containingneutral lipid and CTAB (1:1). However, second generation micelles areable to target tumors in vivo. Weissig and co-workers (1998) used thesoybean trypsin inhibitor (STI) as a model protein to target tumors. STIwas modified with a hydrophobic residue ofN-glutaryl-phosphatidyl-ethanolamine (NGPE) and incorporated into bothpolyethyleneglycol (MW 5000)-distearoyl phosphatidyl ethanolamine(PEG-DSPE) micelles (<20 nm) and PEG-DSPE-modified long-circulatingliposomes (ca. 100 nm). As determined from the protein label by using¹¹¹In attached to soybean trypsin inhibitor via protein-attacheddiethylene triamine pentaacetic acid, DTPA, PEG-lipid micellesaccumulated better than the same protein anchored in long-circulatingPEG-liposomes in subcutaneously established Lewis lung carcinoma in miceafter tail vein injection.

Loading a liposomal dispersion with an amphiphilic drug may cause aphase transformation into a micellar solution. The transition from highratios of phospholipid to drug (from 2:1 to 1:1 downwards) wereaccompanied by the conversion of liposomal dispersions of milky-whiteappearance (particle size 200 nm) to nearly transparent micelles(particle size below 25 nm). See, Schutze and Muller-Goymann (1998).

Fusogenic Peptides

In a further embodiment, the liposome encapsulated DNA described hereinfurther comprises an effective amount of a fusogenic peptide. Fusogenicpeptides belong to a class of helical amphipathic peptides characterizedby a hydrophobicity gradient along the long helical axis. Thishydrophobicity gradient causes the tilted insertion of the peptides inmembranes, thus destabilizing the lipid core and, thereby, enhancingmembrane fusion (Decout et al., 1999).

Hemagglutinin (HA) is a homotrimeric surface glycoprotein of theinfluenza virus. In infection, it induces membrane fusion between viraland endosomal membranes at low pH. Each monomer consists of thereceptor-binding HA1 domain and the membrane-interacting HA2 domain. TheNH₂-terminal region of the HA2 domain (amino acids 1 to 127), theso-called “fusion peptide,” inserts into the target membrane and plays acrucial role in triggering fusion between the viral and endosomalmembranes. Based on the substitution of eight amino acids in region 5-14with cysteines and spin-labeling electron paramagnetic resonance, it wasconcluded that the peptide forms an alphα-helix tilted approximately 25degrees from the horizontal plane of the membrane with a maximum depthof 15 Å from the phosphate group (Macosko et al., 1997). Use offusogenic peptides from influenza virus hemagglutinin HA-2 enhancedgreatly the efficiency of transferrin-polylysine-DNA complex uptake bycells. The peptide was linked to polylysine and the complex wasdelivered by the transferrin receptor-mediated endocytosis (reviewed byBoulikas, 1998a). This peptide has the sequence: GLFEAIAGFI ENGWEGMIDGGGYC (SEQ ID NO:9) and is able to induce the release of the fluorescentdye calcein from liposomes prepared with egg yolk phosphatidylcholine,which was higher at acidic pH. This peptide was also able to increase upto 10-fold the anti-HIV potency of antisense oligonucleotides, at aconcentration of 0.1-1 mM, using CEM-SS lymphocytes in culture. Thispeptide changes conformation at the slightly more acidic environment ofthe endosome, destabilizing and breaking the endosomal membrane(reviewed by Boulikas, 1998a).

The presence of negatively charged lipids in the membrane is importantfor the manifestation of the fusogenic properties of some peptides, butnot of others. Whereas the fusogenic action of a peptide, representing aputative fusion domain of fertilin, a sperm surface protein involved insperm-egg fusion, was dependent upon the presence of negatively chargedlipids, that of the HIV2 peptide was not (Martin and Ruysschaert, 1997).

For example, to analyze the two domains on the fusogenic peptides ofinfluenza virus hemagglutinin HA, HA-chimeras were designed in which thecytoplasmic tail and/or transmembrane domain of HA was replaced with thecorresponding domains of the fusogenic glycoprotein F of Sendai virus.Constructs of HA were made in which the cytoplasmic tail was replaced bypeptides of human neurofibromin type 1 (NF1) (residues 1441 to 1518) orc-Raf-1, (residues 51 to 131) and were expressed in CV-1 cells by usingthe vaccinia virus-T7 polymerase transient-expression system. Membranefusion between CV-1 cells and bound human erythrocytes (RBCs) mediatedby parental or chimeric HA proteins showed that, after the pH waslowered, a flow of the aqueous fluorophore calcein from preloaded RBCsinto the cytoplasm of the protein-expressing CV-1 cells took place. Thisindicated that membrane fusion involves both leaflets of the lipidbilayers and leads to formation of an aqueous fusion pore (Schroth-Diazet al., 1998).

A remarkable discovery was that the TAT protein of HIV is able to crosscell membranes (Green and Loewenstein, 1998) and that a 36-amino aciddomain of TAT, when chemically cross-linked to heterologous proteins,conferred the ability to transduce into cells. The 11-amino acidfusogenic peptide of TAT (YGRKKRRQRRR (SEQ ID NO:10)) is a nucleolarlocalization signal (see Boulikas, 1998b).

Another protein of HIV, the glycoprotein gp41, contains fusogenicpeptides. Linear peptides derived from the membrane proximal region ofthe gp41 ectodomain have potential applications as anti-HIV agents andinhibit infectivity by adopting a helical conformation (Judice et al.,1997). The 23 amino acid residue, N-terminal peptide of HIV-1 gp41 hasthe capacity to destabilize negatively charged large unilamellarvesicles. In the absence of cations, the main structure was apore-forming alphα-helix, whereas in the presence of Ca²⁺ theconformation switched to a fusogenic, predominantly extended beta-typestructure. The fusion activity of HIV(ala) (bearing the R22→Asubstitution) was reduced by 70%, whereas fusogenicity was completelyabolished when a second substitution (V2→E) was included, arguing thatit is not an alpha-helical but an extended structure adopted by theHIV-1 fusion peptide that actively destabilizes cholesterol-containing,electrically neutral membranes (Pereira et al., 1997).

The prion protein (PrP) is a glycoprotein of unknown function normallyfound at the surface of neurons and of glial cells. It is involved indiseases such as bovine spongiform encephalopathy, and Creutzfeldt-Jakobdisease in humans, where PrP is converted into an altered form (termedPrPSc). According to computer modeling calculations, the 120 to 133 and118 to 135 domains of PrP are tilted lipid-associating peptidesinserting in a oblique way into a lipid bilayer and able to interactwith liposomes to induce leakage of encapsulated calcein (Pillot et al.,1997b).

The C-terminal fragments of the Alzheimer amyloid peptide (amino acids29-40 and 29-42) have properties related to those of the fusion peptidesof viral proteins inducing fusion of liposomes in vitro. Theseproperties could mediate a direct interaction of the amyloid peptidewith cell membranes and account for part of the cytotoxicity of theamyloid peptide. In view of the epidemiologic and biochemical linkagesbetween the pathology of Alzheimer's disease and apolipoprotein E (apoE)polymorphism, examination of the potential interaction between the threecommon apoE isoforms and the C-terminal fragments of the amyloid peptideshowed that only apoE2 and apoE3, not apoE4, are potent inhibitors ofthe amyloid peptide fusogenic and aggregational properties. Theprotective effect of apoE against the formation of amyloid aggregateswas thought to be mediated by the formation of stable apoE/amyloidpeptide complexes (Pillot et al., 1997a; Lins et al., 1999).

The fusogenic properties of an amphipathic net-negative peptide (WAE11), consisting of 11 amino acid residues were strongly promoted whenthe peptide was anchored to a liposomal membrane. The fusion activity ofthe peptide appeared to be independent of pH and membrane merging, andthe target membranes required a positive charge that was provided byincorporating lysine-coupled phosphatidylethanolamine (PE-K). Whereasthe coupled peptide could cause vesicle aggregation via nonspecificelectrostatic interaction with PE-K, the free peptide failed to induceaggregation of PE-K vesicles (Pecheur et al., 1997).

A number of studies suggest that stabilization of an alpha-helicalsecondary structure of the peptide after insertion in lipid bilayers inmembranes of cells or liposomes is responsible for the membrane fusionproperties of peptides. Zn²⁺, enhances the fusogenic activity ofpeptides because it stabilizes the alpha-helical structure. For example,the HEXXH (SEQ ID NO:11) domain of the salivary antimicrobial peptide,located in the C-terminal functional domain of histatin-5, a recognizedzinc-binding motif is in a helicoidal conformation (Martin et al., 1999;Melino et al., 1999; Curtain et al., 1999).

Fusion peptides have been formulated with DNA plasmids to createpeptide-based gene delivery systems. A combination of the YKAKnWK (SEQID NO:12) peptide, used to condense plasmids into 40 to 200 nmnanoparticles, with the GLFEALLELLESLWELLLEA (SEQ ID NO:13) amphipathicpeptide, that is a pH-sensitive lytic agent designed to facilitaterelease of the plasmid from endosomes enhanced expression systemscontaining the beta-galactosidase reporter gene (Duguid et al., 1998).See Table 2, below.

TABLE 2 Fusogenic peptides Fusogenic peptide Source Protein PropertiesReference GLFEAIAGFIENGWEG Influenza virus Endowed with membraneBongartz et al., 1994 MIDGGGYC (SEQ ID hemagglutinin fusion propertiesNO: 9) HA-2 YGRKKRRQRRR (SEQ TAT of HIV Endowed with membrane Green andID NO: 5) fusion properties Loewenstein, 1988 23-residue fusogenic N-HIV-1 trans- Was able to insert as an Curtain et al., 1999 terminalpeptide membrane alpha-helix into neutral glycoprotein gp41 phospholipidbilayers 70 residue peptide (SV- Fusion peptide Induced lipid mixing ofegg Ghosh and Shai, 117) and N-terminal phosphatidylcholine- 1999 heptadrepeat of phosphatidyiglycerol Sendai virus (PC/PG) large unilamellarvesicles (LUVs) 23 hydrophobic amino S protein of A high degree ofsimilarity Rodriguez-Crespo et acids in the amino-terminal hepatitis Bvirus with known fusogenic al., 1994 region (HBV) peptides from otherviruses. MSGTFGGILAGLIGLL N-terminal region Was inserted into theRodriguez-Crespo et (SEQ ID NO: 6) of the S protein of hydrophobic coreof the al., 1999 duck hepatitis B lipid bilayer and induced Virus (DHBV)leakage of internal aqueous contents from both neutral and negativelycharged liposomes MSPSSLLGLLAGLQVV S protein of Was inserted into theRodriguez-Crespo et (SEQ ID NO: 14) woodchuck hydrophobic core of theal., 1999 hepatitis B virus lipid bilayer and induced (WHV) leakage ofinternal aqueous contents from both neutral and negatively chargedliposomes N-terminus of Nef Nef protein of Membrane-perturbing andMacreadie et al., human fusogenic activities in 1997 immunodeficiencyartificial membranes; causes type 1 (HIV-1) cell killing in E. coli andyeast Amino-terminal sequence F1 polypeptide of Can be used as a carrierPartidos et al., 1996 F1 polypeptide measles virus system for CTLepitopes (MV) 19-27 amino acid segment Glycoprotein Adopts anamphiphilic Voneche et al., 1992 gp51 of bovine structure and plays akey leukemia virus role in the fusion events induced by bovine leukemiavirus 120 to 133 and 118 to 135 Prion protein Tilted lipid-associatingPillot et al., 1997b domains peptide; interact with liposomes to induceleakage of encapsulated calcein 29-42-residue fragment Alzheimer's beta-Endowed with capacities Lins et al., 1999 amyloid peptide resemblingthose of the tilted fragment of viral fusion proteins Non-aggregatedamyloid Alzheimer's beta- Induces apoptotic neuronal Pillot et al., 1999beta-peptide (1-40) amyloid peptide cell death LCAT 56-68 helicalLecithin Forms stable beta-sheets in Peelman et al., 1999; segmentcholesterol lipids Decout et al., 1999 acyltransferase (LCAT) Peptidesequence B18 Membrane- Triggers fusion between Ulrich et al., 1999associated sea lipid vesicles; a histidine- urchin sperm rich motif forbinding zinc protein binding is required for the fusogenic function53-70 (C-terminal helix) Apolipoprotein Induces fusion of Lambert etal., 1998 (apo) AII unilamellar lipid vesicles and displaces apo AI fromHDL and r-HDL Residues 90-111 PH-30 alpha (a Membrane-fusogenic Niidomeet al., 1997 protein activity to acidic functioning in phospholipidbilayers sperm-egg fusion) Casein signal peptides Alpha s2- and Interactwith Creuzenet et al., 1997 beta-casein dimyristoylphosphatidyl-glycerol and -choline liposomes; show both lytic and fusogenicactivities Pardaxin Amphipathic Forms voltage-gated, Lelkes andpolypeptide, cation-selective pores; Lazarovici, 1988 purified from themediated the aggregation of gland secretion of liposomes composed of theRed Sea phosphatidylserine but not Moses sole of phosphatidylcholineflatfish Pardachirus marmoratus Histatin-5 Salivary Aggregates and fusesMelino et al., 1999 antimicrobial negatively charged small peptideunilamellar vesicles in the presence of Zn2+ Gramicidin (linearAntibiotic Induces aggregation and Massari and Colonna, hydrophobicpolypeptide) fusion of vesicles 1986; Tournois et al., 1990 Amphipathicnegatively Synthetic Forms an alpha-helix Martin et al., 1999 chargedpeptide consisting of inserted and anchored into 11 residues (WAE) themembrane (favored at 37° C.) oriented almost parallel to the lipid acylchains; promotes fusion of large unilamellar liposomes (LUV) A polymerof polylysine Synthetic Histidyl residues become Midoux and (average190) partially cationic upon protonation of Monsigny, 1999 substitutedwith histidyl the imidazole groups at pH residues below 6.0.; disruptendosomal membranes GLFEALLELLESLWELL Synthetic Amphipathic peptide; apH- Duguid et al., 1998 LEA (SEQ ID NO: 4) sensitive lytic agent tofacilitate release of the plasmid from endosomes (LKKL)₄ (SEQ ID NO: 15)Synthetic Amphiphilic fusogenic Gupta and Kothekar, peptide, able tointeract with 1997 four molecules of DMPC Ac-(Leu-Ala-Arg-Leu)₃-Synthetic; basic Caused a leakage of Suenaga et al., 1989; NHCH₃ (SEQ IDNO: 16) amphipathic contents from small Lee et al., 1992 peptidesunilamellar vesicles composed of egg yolk phosphatidylcholine and eggyolk phosphatidic acid (3:1) Amphiphilic anionic Synthetic Can mimic thefusogenic Murata et al., 1991 peptides E5 and E5L activity of influenzahemagglutinin (HA) 30-amino acid peptide with Synthetic; Becomes anamphipathic Parente et al., 1988 the major repeat unit Glu- designed tomimic alpha-helix as the pH is Ala-Leu-Ala (GALA)₇ the behavior oflowered to 5.0; fusion of (SEQ ID NO: 17) the fusogenicphosphatidylcholine small sequences of viral unilamellar vesiclesinduced fusion proteins by GALA requires a peptide length greater than16 amino acids Poly Glu-Aib-Leu-Aib Synthetic Amphiphilic structure uponKono et al., 1993 (SEQ ID NO: 18) Aib the formation of alpha- represents2- helix; caused fusion of aminoisobutyric acid EYPC liposomes anddipalmitoylphosphatidylcholine liposomes more strongly with decreasingpH

Fusogenic Lipids

DOPE is a fusogenic lipid; elastase cleavage ofN-methoxy-succinyl-Ala-Ala-Pro-Val-DOPE (SEQ ID NO:19) converted thisderivative to DOPE (overall positive charge) to deliver an encapsulatedfluorescent probe, calcein, into the cell cytoplasm (Pak et al., 1999).An oligodeoxynucleic sequence of 30 bases complementary to a region ofbeta-endorphin mRNA elicited a concentration-dependent inhibition ofbeta-endorphin production in cell culture after it was encapsulatedwithin small unilamellar vesicles (50 nm) containingdipalmitoyl-DL-alpha-phosphatidyl-L-serine endowed with fusogenicproperties (Fresta et al., 1998).

Nuclear Localization Signals (NLS)

In a further embodiment, the liposome encapsulated plasmid oroligonucleotide DNA described herein further comprise an effectiveamount of nuclear localization signal (NLS) peptides. Trafficking ofnuclear proteins from the site of their synthesis in the cytoplasm tothe sites of function in the nucleus through pore complexes is mediatedby NLSs on proteins to be imported into nuclei (Tables 3-10, below).Protein translocation from the cytoplasm to the nucleoplasm involves:(i) the formation of a complex of karyopherin a with NLS-protein; (ii)subsequent binding of karyopherin β; (iii) binding of the complex toFXFG peptide repeats on nucleoporins; (iv) docking of Ran-GDP tonucleoporin and to karyopherin heterodimer by p10; (v) a number ofassociation-dissociation reactions on nucleoporins that dock the importsubstrate toward the nucleoplasmic side with a concomitant GDP-GTPexchange reaction transforming Ran-GDP into Ran-GTP and catalyzed bykaryopherin α; and (vi) dissociation from karyopherin and release of thekaryopherin α/NLS-protein by Ran-GTP to the nucleoplasm.

Karyophilic and acidic clusters were found in most non-membraneserine/threonine protein kinases whose primary structure has beenexamined (Table 6). These karyophilic clusters might mediate theanchoring of the kinase molecules to transporter proteins for theirregulated nuclear import and might constitute the nuclear localizationsignals. In contrast to protein transcription factors that areexclusively nuclear possessing strong karyophilic peptides composed ofat least four arginines, (R), and lysines, (K), within an hexapeptideflanked by proline and glycine helix-breakers, protein kinases oftencontain one histidine and three K+R residues (Boulikas, 1996). This wasproposed to specify a weak NLS structure resulting in the nuclear importof a fraction of the total cytoplasmic kinase molecules, as well as intheir weak retention in the different ionic strength nuclearenvironment. Putative NLS peptides in protein kinases may also containhydrophobic or bulky aromatic amino acids proposed to further diminishtheir capacity to act as strong NLS.

Most mammalian proteins that participate in DNA repair pathways seem topossess strong karyophilic clusters containing at least four R+K over astretch of six amino acids (Table 7).

Rules to Predict Nuclear Localization of an Unknown Protein

Several simple rules have been proposed for the prediction of thenuclear localization of a protein of an unknown function from its aminoacid sequence:

(i) An NLS is defined as four arginines (R) plus lysines (K) within anhexapeptide; the presence of one or more histidines (H) in the tetrad ofthe karyophilic hexapeptide, often found in protein kinases that have acytoplasmic and a nuclear function, may specify a weak NLS whosefunction might be regulated by phosphorylation or may specify proteinsthat function in both the cytoplasm and the nucleus (Boulikas, 1996);

(ii) The K/R clusters are flanked by the α-helix breakers G and P thusplacing the NLS at a helix-turn-helix or end of a α-helix.Negatively-charged amino acids (D, E) are often found at the flank ofthe NLS and on some occasions may interrupt the positively-charged NLScluster;

(iii) Bulky amino acids (W, F, Y) are not present within the NLShexapeptide;

(iv) NLS signals may not be flanked by long stretches of hydrophobicamino acids (e.g. five); a mixture of charged and hydrophobic aminoacids serves as a mitochondrial targeting signal;

(v) The higher the number of NLSs, the more readily a molecule isimported to the nucleus (Dworetzky et al., 1988). Even small proteins,for example histones (10-22 kDa), need to be actively imported toincrease their import rates compared with the slow rate of diffusion ofsmall molecules through pores;

(vi) Signal peptides are stronger determinants than NLSs for proteintrafficking. Signal peptides direct proteins to the lumen of theendoplasmic reticulum for their secretion or insertion into cellularmembranes (presence of transmembrane domains) (Boulikas, 1994);

(vii) Signals for the mitochondrial import of proteins (a mixture ofhydrophobic and karyophilic amino acids) may antagonize nuclear importsignals and proteins possessing both type of signals may be translocatedto both mitochondria and nuclei;

(viii) Strong association of a protein with large cytoplasmic structures(membrane proteins, intermediate filaments) make such proteinsunavailable for import even though they posses NLS-like peptides(Boulikas, 1994);

(ix). Transcription factors and other nuclear proteins posses a greatdifferent number of putative NLS stretches. Of the sixteen possibleforms of putative NLS structures the most abundant types are the θθ×θθ,θθθ×θ, θθθθ, and θθ×θ×θ, where θ is R or K, together accounting forabout 70% of all karyophilic clusters on transcription factors(Boulikas, 1994);

(x) A small number of nuclear proteins seem to be void of a typicalkaryophilic NLS. Either non karyophilic peptides function for theirnuclear import, as such molecules possess bipartite NLSs, or theseNLS-less proteins depend absolutely for import on their strongcomplexation in the cytoplasm with a nuclear protein partner able to beimported (Boulikas, 1994). This mechanism may ensure a certainstoichiometric ratio of the two molecules in the nucleus, and might beof physiological significance; and

(xi) A number of proteins may be imported via other mechanisms notdependent on classical NLS.

A number of processes have been found to be regulated by nuclear importincluding nuclear translocation of the transcription factors NF-κB,rNFIL-6, ISGF3, SRF, c-Fos, GR as well as human cyclins A and B1, caseinkinase II, cAMP-dependent protein kinase II, protein kinase C, ERK1 andERK2. Failure of cells to import specific proteins into nuclei can leadto carcinogenesis. For example, BRCA1 is mainly localized in thecytoplasm in breast and ovarian cancer cells, whereas in normal cellsthe protein is nuclear. mRNA is exported through the same route as acomplex with nuclear proteins possessing nuclear export signals (NES).The majority of proteins with NES are RNA-binding proteins that bind toand escort RNAs to the cytoplasm. However, other proteins with NESfunction in the export of proteins; CRM1, that binds to the NES sequenceon other proteins and interacts with the nuclear pore complex, is anessential mediator of the NES-dependent nuclear export of proteins ineukaryotic cells. Nuclear localization and export signals (NLS and NES)are found on a number of important molecules, including p53, v-Rel, thetranscription factor NF-ATc, the c-Abl nonreceptor tyrosine kinase, andthe fragile X syndrome mental retardation gene product. The deregulationof their normal import/export trafficking has important implications forhuman disease. Both nuclear import and export processes can bemanipulated by conjugation of proteins with NLS or NES peptides. Duringgene therapy, the foreign DNA needs to enter nuclei for itstranscription. A pathway is proposed involving the complexation ofplasmids and oligonucleotides with nascent nuclear proteins possessingNLSs as a prerequisite for their nuclear import. Covalent linkage of NLSpeptides to oligonucleotides and plasmids or formation of complexes ofplasmids with proteins possessing multiple NLS peptides was proposed(Boulikas, 1998b) to increase their import rates and the efficiency ofgene expression. Cancer cells were predicted to import more efficientlyforeign DNA into nuclei, compared with terminally differentiated cellsbecause of their increased rates of proliferation and protein import.

Antineoplastic Drugs

In a further embodiment, the liposome encapsulated plasmid oroligonucleotide DNA described herein, further comprises its use forreducing tumor size or restricting its growth with combination withencapsulated or free antineoplastic agents. Antineoplastic agentspreferably are: (i) alkylating agents having thebis-(2-chloroethyl)-amine group such as chlormethine, chlorambucile,melphalan, uramustine, mannomustine, extramustinephosphat,mechlorethaminoxide, cyclophosphamide, ifosfamide, or trifosfamide; (ii)alkylating agents having a substituted aziridine group, for exampletretamine, thiotepa, triaziquone, or mitomycine; (iii) alkylating agentsof the methanesulfonic ester type such as busulfane; (iv) alkylatingN-alkyl-N-nitrosourea derivatives, for example carmustine, lomustine,semustine, or streptozotocine; (v) alkylating agents of themitobronitole, dacarbazine, or procarbazine type; (vi) complexing agentssuch as cis-platin; (vii) antimetabolites of the folic acid type, forexample methotrexate; (viii) purine derivatives such as mercaptopurine,thioguanine, azathioprine, tiamiprine, vidarabine, or puromycine andpurine nucleoside phosphorylase inhibitors; (ix) pyrimidine derivatives,for example fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine,flucytosine; (x) antibiotics such as dactinomycin, daunorubicin,doxorubicin, mithramycin, bleomycin or etoposide; (xi) vinca alkaloids;(xii) inhibitors of proteins overexpressed in cancer cells such astelomerase inhibitors, glutathione inhibitors, proteasome inhibitors;(xiii) modulators or inhibitors of signal transduction pathways such asphosphatase inhibitors, protein kinase C inhibitors, casein kinaseinhibitors, insulin-like growth factor-1 receptor inhibitor, rasinhibitors, ras-GAP inhibitor, protein tyrosine phosphatase inhibitors;(xiv) tumor angiogenesis inhibitors such as angiostatin, oncostatin,endostatin, thalidomide; (xv) modulators of the immune response andcytokines such as interferons, interleukins, TNF-alpha; (xvi) modulatorsof the extracellular matrix such as matrix metalloproteinase inhibitors,stromelysin inhibitors, plasminogen activator inhibitor; (xvii) hormonemodulators for hormone-dependent cancers (breast cancer, prostatecancer) such as antiandrogen, estrogens; (xviii) apoptosis regulators;(xix) bFGF inhibitor; (xx) multiple drug resistance gene inhibitor;(xxi) monoclonal antibodies or antibody fragments against antigenesoverexpressed in cancer cells (anti-Her2/neu for breast cancer); (xxii)anticancer genes whose expression will cause apoptosis, arrest the cellcycle, induce an immune response against cancer cells, inhibit tumorangiogenesis i.e. formation of blood vessels, tumor suppressor genes(p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax, bcl-xs, E2F, IGF-IVEGF, angiostatin, oncostatin, endostatin, GM-CSF, IL-12, IL-2, IL-4,IL-7, IFN-γ, and TNF-α); and (xxiii) antisense oligonucleotides(antisense c-fos, c-myc, K-ras). Optionally these drugs are administeredin combination with chlormethamine, prednisolone, prednisone, orprocarbazine or combined with radiation therapy. Future new anticancerdrugs added to the arsenal are expected to be ribozymes, triplex-formingoligonucleotides, gene inactivating oligonucleotides, a number of newgenes directed against genes that control the cell proliferation orsignaling pathways, and compounds that block signal transduction.

Anti-cancer drugs include: acivicin, aclarubicin, acodazolehydrochloride, acronine, adozelesin, adriamycin, aldesleukin,altretamine, ambomycin, ametantrone acetate, aminoglutethimide,amsacrine, anastrozole, anthramycin, asparaginase, asperlin,azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide,bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleomycinsulfate, brequinar sodium, bropirimine, busulfan, cactinomycin,calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicinhydrochloride, carzelesin, cedefingol, chlorambucil, cirolemycin,cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine,dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine,dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel,doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifenecitrate, dromostanolone propionate, duazomycin, edatrexate, eflornithinehydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine,epirubicin hydrochloride, erbulozole, esorubicin hydrochloride,estramustine, estramustine phosphate sodium, etanidazole, etoposide,etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine,fenretinide, floxuridine, fludarabine phosphate, fluorouracil,fluorocitabine, fosquidone, fostriecin sodium, gemcitabine, gemcitabinehydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide,ilmofosine, interferon alfa-2a, interferon α-2b, interferon α-n1,interferon α-n3, interferon β-i a, interferon γ-i b, iproplatin,irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolideacetate, liarozole hydrochloride, lometrexol sodium, lomustine,losoxantrone hydrochloride, masoprocol, maytansine, mechlorethaminehydrochloride, megestrol acetate, melengestrol acetate, melphalan,menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine,meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin,mitomycin, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolicacid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel,pegaspargase, peliomycin, pentamustine, peplomycin sulfate,perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,prednisone, procarbazine hydrochloride, puromycin, puromycinhydrochloride, pyrazofurin, riboprine, rogletimide, safingol, safingolhydrochloride, semustine, simtrazene, sparfosate sodium, sparsomycin,spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin,streptozocin, sulofenur, talisomycin, taxol, tecogalan sodium, tegafur,teloxantrone hydrochloride, temoporfin, teniposide, teroxirone,testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin,tirapazamine, topotecan hydrochloride, toremifene citrate, trestoloneacetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate,triptorelin, tubulozole hydrochloride, uracil mustard, uredepa,vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate,vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate,vinleurosine sulfate, vinorelbine tartrate, vinzolidine sulfate,vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicinhydrochloride.

Other anti-cancer drugs include: 20-epi-1,25 dihydroxyvitamin D3,5-ethynyluracil, abiraterone, aclarubicin, acylfulvene, adecypenol,adozelesin, aldesleukin, ALL-TK antagonists, altretamine, ambamustine,amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine,anagrelide, anastrozole, andrographolide, angiogenesis inhibitors,antagonist D, antagonist G, antarelix, anti-dorsalizing morphogeneticprotein-1, anti androgen, antiestrogen, antineoplaston, antisenseoligonucleotides, aphidicolin glycinate, apoptosis gene modulators,apoptosis regulators, apurinic acid, ara-CDP-DL-PTBA, argininedeaminase, asulacrine, atamestane, atrimustine, axinastatin 1,axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatinIII derivatives, balanol, batimastat, BCR/ABL antagonists,benzochlorins, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor,bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistrateneA, bizelesin, breflate, bropirimine, budotitane, buthionine sulfoximine,calcipotriol, calphostin C, camptothecin derivatives, canarypox IL-2,capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, CaRestM3, CARN 700, cartilage derived inhibitor, carzelesin, casein kinaseinhibitors (ICOS), castanospermine, cecropin B, cetrorelix, chlorins,chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine,clomifene analogues, clotrimazole, collismycin A, collismycin B,combretastatin A4, combretastatin analogue, conagenin, crambescidin 816,crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A,cyclopentanthraquinones, cycloplatam, cypemycin, cytarabine ocfosfate,cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B,deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone,didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine,dihydrotaxol, 9-dioxamycin, diphenyl spiromustine, docosanol,dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA,ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene,emitefur, epirubicin, epristeride, estramustine analogue, estrogenagonists, estrogen antagonists, etanidazole, etoposide phosphate,exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride,flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunorunicinhydrochloride, forfenimex, formestane, fostriecin, fotemustine,gadolinium gallium nitrate texaphyrin, galocitabine, ganirelix,gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam,heregulin, hexamethylene bisacetamide, hypericin, ibandronic acid,idarubicin, idoxifene, idramantone, ilmofosine, ilomastat,imidazoacridones, imiquimod, immunostimulant peptides, insulin-likegrowth factor-1 receptor inhibitor, interferon agonists, interferons,interleukins, iobenguane, iododoxorubicin, ipomeanol, 4-, irinotecan,iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole,leukemia inhibiting factor, leukocyte alpha interferon,leuprolide+estrogen+progesterone, leuprorelin, levamisole, liarozole,linear polyamine analogue, lipophilic disaccharide peptide, lipophilicplatinum compounds, lissoclinamide 7, lobaplatin, lombricine,lometrexol, lonidamine, losoxantrone, lovastatin, loxoribine,lurtotecan, lutetium texaphyrin, lysofylline, lytic peptides,maitansine, mannostatin A, marimastat, masoprocol, maspin, matrilysininhibitors, matrix metalloproteinase inhibitors, menogaril, merbarone,meterelin, methioninase, metoclopramide, MIF inhibitor, mifepristone,miltefosine, mirimostim, mismatched double stranded RNA, mitoguazone,mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mofarotene, molgramostim, monoclonalantibody, human chorionic gonadotrophin, monophosphoryl lipidA+myobacterium cell wall sk, mopidamol, multiple drug resistance geneinhibitor, multiple tumor suppressor 1-based therapy, mustard anticanceragent, mycaperoxide B, mycobacterial cell wall extract, myriaporone,N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip,naloxone+pentazocine, napavin, naphterpin, nartograstim, nedaplatin,nemorubicin, neridronic acid, neutral endopeptidase, nilutamide,nisamycin, nitric oxide modulators, nitroxide antioxidant, nitrullyn,O6-benzylguanine, octreoti de, okicenone, oligonucleotides, onapristone,ondansetron, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, paclitaxel analogues, paclitaxelderivatives, palauamine, palmitoylrhizoxin, pamidronic acid,panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase,peldesine, pentosan polysulfate sodium, pentostatin, pentrozole,perflubron, perfosfamide, perillyl alcohol, phenazinomycin,phenylacetate, phosphatase inhibitors, picibanil, pilocarpinehydrochloride, pirarubicin, piritrexim, placetin A, placetin B,plasminogen activator inhibitor, platinum complex, platinum compounds,platinum-triamine complex, porfimer sodium, porfiromycin, propylbis-acridone, prostaglandin J2, proteasome inhibitors, protein A-basedimmune modulator, protein kinase C inhibitor, protein kinase Cinhibitors, microalgal., protein tyrosine phosphatase inhibitors, purinenucleoside phosphorylase inhibitors, purpurins, pyrazoloacridine,pyridoxylated hemoglobin polyoxyethylene conjugate, raf antagonists,raltitrexed, ramosetron, ras farnesyl protein transferase inhibitors,ras inhibitors, ras-GAP inhibitor, retelliptine demethylated, rhenium Re186 etidronate, rhizoxin, ribozymes, RH retinamide, rogletimide,rohitukine, romurtide, roquinimex, rubiginone B1, ruboxyl, safingol,saintopin, SarCNU, sarcophytol A, sargramostim, Sdi 1 mimetics,semustine, senescence derived inhibitor 1, sense oligonucleotides,signal transduction inhibitors, signal transduction modulators, singlechain antigen binding protein, sizofuran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosic acid, spicamycin D, spiromustine,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, stromelysin inhibitors, sulfinosine,superactive vasoactive intestinal peptide antagonist, suradista,suramin, swainsonine, synthetic glycosaminoglycans, tallimustine,tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium,tegafur, tellurapyrylium, telomerase inhibitors, temoporfin,temozolomide, teniposide, tetrachlorodecaoxide, tetrazomine,thaliblastine, thalidomide, thiocoraline, thrombopoietin, thrombopoietinmimetic, thymalfasin, thymopoietin receptor agonist, thymotrinan,thyroid stimulating hormone, tin ethyl etiopurpurin, tirapazamine,titanocene dichloride, topotecan, topsentin, toremifene, totipotent stemcell factor, translation inhibitors, tretinoin, triacetyluridine,triciribine, trimetrexate, triptorelin, tropisetron, turosteride,tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex,urogenital sinus-derived growth inhibitory factor, urokinase receptorantagonists, vapreotide, variolin B, velaresol, veramine, verdins,verteporfin, vinorelbine, vinxaltine, vitaxin, vorozole, zanoterone,zeniplatin, zilascorb, zinostatin stimalamer.

pH-Sensitive Peptide-DNA Complexes

In a further embodiment of the invention, the genes in plasmid DNA arebrought in interaction with fusogenic peptide/NLS conjugates. In afurther embodiment the NLS moiety is a stretch of histidyl residues ableto assume a net positive charge at a pH of about 5 to 6 and to show areduction or loose completely this charge at pH above 7. Theelectrostatic interaction of these positively-charged peptides with thenegatively-charged plasmid DNA molecules, established at pH 5-6 isweakened at physiological pH (pH-sensitive peptide-DNA complexes).

The first step of the present invention involves complex formationbetween the plasmid or oligonucleotide DNA with the histidyl/fusogenicpeptide conjugate and lipid components in 10-90% ethanol at pH 5.0 to6.0. The conditions must be where the histidyl residues have a netpositive charge and can establish electrostatic interactions withplasmids, oligonucleotides or negatively-charged drugs. At the sametime, the presence of the positively-charged lipid molecules promotesformation of micelles. At the second step, micelles are converted intoliposomes by dilution with water and mixing with pre-made liposomes orlipids at pH 5-6. This is followed by dialysis against pH 7 andextrusion through membranes, entrapping and encapsulating plasmids oroligonucleotides to with a very high yield.

Whereas the composition of peptides and cationic lipids in the firststep provides the lipids of the internal bilayer, the type of liposomesor lipids added at step 2 provide the external coating of the finalliposome formulation (FIG. 1). Examples for the formulations of peptidesinclude: HHHHHSPSL₁₆ (SEQ ID NO:623), and HHHHHSPS(LAI)₅ (SEQ IDNO:624).

These are added at a 1:0.5:0.5 molar ratio (negative charge on DNA:cationic liposome: histidine peptide). The peptide inserts in analpha-helical conformation inside the lipid bilayer and not only carriesout DNA condensation but also endows membrane fusion properties to thecomplex to improve entrance across the cell membrane. The type ofhydrophobic amino acids (for example, content in aromatic amino acids),in the peptide chain is very important as is the length of the peptidechain in ensuring integrity and rigidity of the complexes. Coating theouter surface of the complexes with polyethyleneglycol, hyaluronic acidsand other polymers conjugated to lipids gives the particles longcirculation properties in body fluids and the ability to target solidtumors and their metastases after intravenous injection, and also theability to cross the tumor cell membrane.

Protease-Sensitive Linkages in Peptides Between the NLS and FusogenicMoieties

Conversion of Micelles into Liposomes

An important issue of the present invention is the conversion ofmicelles formed between the DNA and the cationic lipids, in the presenceof ethanol, into liposomes. This is done by the direct addition of themicelle complex into an aqueous solution of preformed liposomes. Theliposomes have an average size of 80-160 nm or vice versa, leading to asolution of a final ethanol concentration below 10%. A formulationsuitable for pharmaceutical use and for injection into humans andanimals will require that the liposomes are of neutral composition (suchas cholesterol, PE, PC) coated with PEG.

However, another important aspect is the research application of thepresent invention, such as for transfection of cells in culture. Thecomposition of the aqueous solution of liposomes is any type ofliposomes containing cationic lipids and suitable therefore fortransfection of cells in culture such as DDAB:DOPE 1:1. These liposomesare pre-formed and downsized by sonication or extrusion throughmembranes to a diameter of 80-160 nm. The ethanolic micelle preparationsare then added to the aqueous solution of liposomes with a concomitantdilution of the ethanol solution to below 10%. This step will result infurther condensation of DNA or interaction of the negatively-chargedphosphate groups on DNA with positively charged groups on lipids. Caremust be taken so as only part of the negative charges on DNA areneutralized by lipids in the micelle. The remaining chargeneutralization of the DNA is to be provided by the cationic component ofthe preformed liposomes in the second step.

Regulatory DNA and Nuclear Matrix-Attached DNA

In a further embodiment of the present invention, the genes in plasmidDNA are driven by regulatory DNA sequences isolated from nuclearmatrix-attached DNA using shotgun selection approaches.

The compact structural organization of chromatin and the proper spatialorientation of individual chromosomes within a cell are partiallyprovided by the nuclear matrix. The nuclear matrix is composed of DNA,RNA and proteins and serves as the site of DNA replication, genetranscription, DNA repair, and chromosomal attachment in the nucleus.Diverse sets of DNA sequences have been found associated with nuclearmatrices and is referred to as matrix attachment regions or MARs. TheMARs serve many functions, acting as activators of gene transcription,silencers of gene expression, insulators of transcriptional activity,nuclear retention signals and origins of DNA replication. Currentstudies indicate that different subsets of MARs are found in differenttissue types and may assist in regulating the specific functions ofcells. The presence of this complex assortment of structural andregulatory molecules in the matrix, as well as the in situ localizationof DNA replication and transcription complexes to the matrix stronglysuggest that the nuclear matrix plays a fundamental, unique role innuclear processes. The structuring of genomes into domains has afunctional significance. The inclusion of specific MAR elements withingene transfer vectors could have utility in many experimental and genetherapy applications. Many gene therapy applications require specificexpression of one or more genes in targeted cell types for prolongedtime periods. MARs within vectors could enhance transcription of theintroduced transgene, prolong the retention of that sequence within thenucleus or insulate expression of that transgene from the expression ofa cotransduced gene (reviewed by Boulikas, 1995; Bode et al, 1996).

Various biochemical procedures have been used to identify regulatoryregions within genes. Traditionally, identification and selection ofregulatory DNA sequences depend on tedious procedures such astranscription factor footprinting in vitro or in vivo, or subcloning ofsmaller fragments from larger genomic DNA sequences upstream of reportergenes. These methods have been used primarily to identify regionsproximal to the 5′ end of genes. However, in many instances, regulatoryregions are found at considerable distances from the proximal 5′ end ofthe gene, and confer cell type- or developmental stage-specificity. Forexample, studies from the groups of Grosveld and Engel (Lakshmanan etal., 1999) have shown that over 625 kb of genomic sequences surroundingthe GATA-3 locus are required for the correct developmental expressionof the gene in transgenic mice. Extensive DNA stretches at distances5-20 kb upstream of the gene were found to be responsible for thecentral nervous system-specificity of expression. The region between 20to 130 kb upstream of the gene harbored regulatory regions forurogenital-specific expression of GATA-3, whereas sequences 90-180 kbdownstream of the gene conferred endocardial-specific expression.

The presently disclosed method has the potential of rapidly identifyingregulatory control regions. In cells, chromatin loops are formed anddifferent attachment regions are used in different cell types or stagesof development to modulate the expression of a gene. The presentlydisclosed method for isolating regulatory regions based on theirattachment to the nuclear matrix can identify regulatory regionsirrespective of their distance from the gene. Although the human genomeproject is expected to be almost complete by the year 2000, informationon the location and nature of the vast majority of the estimated 500,000regulatory regions will not be available.

Example 1

Plasmid DNA condenses with various agents, as well as variousformulations of cationic liposomes. The condensation affects the levelof expression of the reporter beta-galactosidase gene after transfectionof K562 human erythroleukemia cell cultures. Liposome compositions areshown in the Table below and in FIG. 2. All lipids were from AvantiPolar Lipids (700 Industrial Park Drive, Alabaster, Ala. 35007). Theoptimal ratio of lipid to DNA was 7 nmoles total lipid/μg DNA. Thetransfection reagent (10 μg DNA mixed with 70 nmoles total lipid) wastransferred to a small culture flask followed by the addition of 10 mlK562 cell culture (about 2 million cells total); mixing of cells withthe transfection reagent was at 5-10 min after mixing DNA withliposomes. Cells were assayed for beta-galactosidase activity severaltimes at 1-30 days post-transfection. The transfected cells weremaintained in cell culture as normal cell cultures.

Best results were obtained when the cells used for transfection were atlow number, not near confluence. In all experiments the transfectionmaterial was added directly in the presence of serum and antibioticswithout removal of the transfection reagent or washings of the cells.This simplifies the transfection procedure and is suitable for lymphoidand other type of cell cultures that do not attach to the dish, but growin suspension. All DNA condensing agents were purchased from Sigma. Theywere suspended at 0.1 mg/ml in water. Plasmid pCMVβ was purchased fromClontech and was purified using the Anaconda kit of Althea Technologies(San Diego, Calif.). PolyK is polylysine, mw 9,400. PolyR ispolyarginine. PolyH is polyhistidine.

To 100 μl plasmid solution (10 μg total plasmid DNA) 20 μl or 50 μl ofpolyK, polyR, polyH, were added; the volume was adjusted to 250 μl withwater followed by addition of about 70 μl liposomes (7 nmoles 4 μg DNA).After incubation for 10 min to 1 h at 20° C. the transfection mixturewas brought in contact with the cell culture. The best DNA condensingreagent was polyhistidine compared with the popular polylysine. The bestcationic lipid was DC-cholesterol (DC-CHOL:3β[N—(N′,N′-dimethylaminoethane)carbamoyl]cholesterol). SFV is SemlikiForest virus expressing beta-galactosidase. The results are shown inFIG. 2.

Liposome Molecular weight Composition Preparation L2 DDAB mw 631 DDAB4.2 μmoles/ml 15 mg DDAB + 0.88 ml DOPE mw 744 DOPE 4.2 μmoles/ml 20mg/ml DOPE L3 DOGS-NTA DOGS-NTA 1 μmole/ml 5 mg DOGS mw 1015.4 DOPE 1μmole/ml 0.185 ml DOPE L4 DC-Chol (mw 537) DC-Chol 1 μmole/ml 0.106 mlDC-Chol DOPE (mw 744) DOPE 1 μmole/ml (25 mg/ml) + 0.185 ml DOPE (20mg/ml) L5 DOTAP (mw 698) DOTAP 1.4 μmole/ml 0.5 ml 10 mg/ml DOTAP + DOPE(mw 744) DOPE 1.3 μmole/ml 0.25 ml DOPE (20 mg/ml) L6 DODAP (mw 648)DODAP 1.54 μmoles/ml 0.5 ml 10 mg/ml DODAP = DOPE 1.3 μmole/ml 5 mg =7.72 μmoles + 0.25 ml DOPE (20 mg/ml)

Example 2 Targeting Genes to Tumors Using Gene Vehicles (Lipogenes)

As shown in FIG. 3, tumor targeting in SCID (severe combinedimmunodeficient) mice were implanted subcutaneously, at two sites, withhuman MCF-7 breast cancer cells. The cells were allowed to develop intolarge, measurable solid tumors at about 30 days post-inoculation. Micewere injected intraperitoneously with 0.2 mg plasmid pCMVβ DNA (size ofthe plasmid is ˜4 kb) per animal carrying the bacterialbeta-galactosidase reporter gene. Plasmid DNA (200 μg, 2.0 mg/ml, 0.1ml) was incubated for 5 min with 200 μl neutral liposomes of thecomposition 40% cholesterol, 20% dioleoylphosphatidylethanolamine(DOPE),12% palmitoyloleoylphosphatidylcholine (POPC), 10% hydrogenated soyphosphatidylcholine (HSPC), 10% distearoylphosphatidylethanolamine(DSPE), 5% sphingomyelin (SM), and 3% derivatized vesicle-forming lipidM-PEG-DSPE.

At this stage, weak complexation of plasmid DNA with neutral(zwitterionic) liposomes takes place. This ensures homogeneousdistribution of plasmid DNA to liposomes at the subsequent step ofaddition of cationic liposomes. After complexation of plasmid DNA withzwitterionic liposomes, 50 μl of cationic liposomes (DC-Chol 1mmole/ml:DOPE 1.4 mmole/ml) were added and incubated at room temperaturefor 10 min. At this stage, a mixed liposome population is present and,most likely, formation of a type of liposome-DNA complexes containinglipids from the zwitterionic and cationic lipids takes place. Thematerial was injected (0.35 ml total volume) to the intraperitonealcavity of the animal. At 5 days post-injection the animal wassacrificed, the skin was removed and the carcass was incubated intoX-gal staining solution for about 30 min at 37° C. The animal wasincubated in fixative in X-gal staining for about 30 min (addition of100 μl concentrated glutaraldehyde to 30 ml X-gal staining solution) andthe incubation in staining solution continued. Photos were taken in atime course during the incubation period revealing the preferred organswhere beta-galactosidase expression took place.

Because of the tumor vasculature targeting shown in FIG. 3E, the dataimply that transfer of the genes of angiostatin, endostatin, oroncostatin to the tumors (whose gene products restrict vascular growthand inhibit blood supply to the tumor) is expected to be a rationalapproach for cancer treatment. Also, a combination therapy usinganticancer lipogenes with encapsulated drugs into tumor targetingliposomes appears as a rational cancer therapy.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand the following examples are intended to illustrate and not limit thescope of the invention. Other aspects, advantages and modificationswithin the scope of the invention will be apparent to those skilled inthe art to which the invention pertains.

TABLE 3 Simple NLS Signal oligopeptide Protein and features PKKKRKV (SEQID NO: 20) Wild-type SV40 large T protein A point mutation convertinglysine-128 (double underlined) to threonine results in the retention oflarge T in the cytoplasm. Transfer of this peptide to the N-terminus ofβ-galactosidase or pyruvate kinase at the gene level and microinjectionof plasmids into Vero cells showed nuclear location of chimericproteins. PKKKRMV (SEQ ID NO: 21) SV40 large T with a K→M change.Site-directed mutagenesis only slightly impaired nuclear import of largeT. PKKKRKVEDP (SEQ ID Synthetic NLS peptide from SV40 large T antigencrosslinked to BSA NO: 22) or IgG mediated their nuclear localizationafter microinjection in Xenopus oocytes. The PKKGSKKA from Xenopus H2Bwas ineffective and PKTKRKV was less effective. CGYGPKKKRKVGG (SEQ IDSynthetic peptide from SV40 large T antigen conjugated to various NO:23) proteins and microinjected into the cytoplasm of TC-7 cells.Specified nuclear localization up to protein sizes of 465 kD (ferritin).IgM of 970 kD and with an estimated radius of 25-40 nm was retained inthe cytoplasm. CYDDEATADSQHSTPPKKK SV40 large T protein long NLS. Thelong NLS but not the short NLS, RKVEDPKDFESELLS was able to localize thebulky IgM (970 kD) into the nucleus. (SEQ ID NO: 24) Mutagenesis at thefour possible sites of phosphorylation (double underlined) impairednuclear import. CGGPKKKRKVG SV40 large T protein. This synthetic peptidecrosslinked to chicken (SEQ ID NO: 25) serum albumin and microinjectedinto HeLa cells caused nuclear localization. PKKKIKV (SEQ ID NO: 26) Amutated (R→I) version of SV40 large T NLS. Effective NLS.MKx₁₁CRLKKLKCSKEKPKC Yeast GAL4 (99 kD). Fusions of the GAL4 geneportion encoding the AKCLKx₅Rx₃KTKR (SEQ ID 74 N-terminal amino acidwith E. coli β-galactosidase introduced into NO: 27) yeast cells specifynuclear localization. 74 N-terminal amino acid MKx₁₁CRLKKLKCSKEKPKCYeast GAL4. Acted as an efficient nuclear localization sequence when A(SEQ ID NO: 28) fused to invertase but not to β-galactosidase introducedby 29 N-terminal amino acid transformation into yeast cells. PKKARED(SEQ ID NO: 29) Polyoma large T protein. Identified by fusion withpyruvate kinase VSRKRPR (SEQ ID NO: 30) cDNA and microinjection of VeroAfrican green monkey cells. Mutually independent NLS. Can exertcooperative effects. CGYGVSRKRPRPG Polyoma virus large T protein. Thissynthetic peptide crosslinked to (SEQ ID NO: 31) chicken serum albuminand microinjected into HeLa cells caused nuclear localization. APTKRKGSSV40 VP1 capsid polypeptide (46 kD). NLS (N terminus) determined (SEQ IDNO: 32) by infection of monkey kidney cells with a fusion constructcontaining the 5′ terminal portion of SV40 VP1 gene and the completecDNA sequence of poliovirus capsid VP1 replacing the VP1 gene of SV40.APKRKSGVSKC (1-11) Polyoma virus major capsid protein VP1 (11 N-terminalamino acid). (SEQ ID NO: 33) Yeast expression vectors coding for 17N-terminal amino acid of VP1 fused to β-galactosidase gave a proteinthat was transported to the nucleus in yeast cells. Subtractiveconstructs of VP1 lacking A¹ to C¹¹ were cytoplasmic. This,FITC-labeled, synthetic peptide crosslinked to BSA or IgG, causednuclear import after microinjection into 3T6 cells. Replacement of K³with T did not. PNKKKRK (SEQ ID NO: 34) SV40 VP2 capsid protein (39 kD).The 3′ end of the SV40 VP2-VP3 (amino acid position 317-323) genescontaining this peptide when fused to poliovirus VP1 capsid protein atthe gene level resulted in nuclear import of the hybrid VP1 in simiancells infected with the hybrid SV40. EEDGPQKKKRRL (307-318) Polyomavirus capsid protein VP2. A construct having truncated VP2 (SEQ ID NO:35) lacking the 307-318 peptide transfected into COS-7 cells showedcytoplasmic retention of VP2. The 307-318 peptide crosslinked to BSA orIgG specified nuclear import following their microinjection into NIH 3T6cells. GKKRSKA (SEQ ID NO: 36) Yeast histone H2B. This peptide specifiednuclear import when fused to β-galactosidase. KRPRP (SEQ ID NO: 37)Adenovirus E1a. This pentapeptide, when linked to the C-terminus of E.coli galactokinase, was sufficient to direct its nuclear accumulationafter microinjection in Vero monkey cells. CGGLSSKRPRP (SEQ IDAdenovirus type 2/5 E1a. This synthetic peptide crosslinked to chickenNO: 38) bovine albumin and microinjected into HeLa cells caused nuclearlocalization. LVRKKRKTE₃SP (NLS 1) Xenopus N1 (590 amino acid). Abundantin X. laevis oocytes, forming (SEQ ID NO: 39) complexes with histonesH3, H4 via two acidic domains each LKDKDAKKSKQE (NLS2) containing 21 and9 (D + E), respectively. The NLS1 is required but not (SEQ ID NO: 40)sufficient for nuclear accumulation of protein N1. NLS 1 and 2 arecontiguous at the C-terminus. GNKAKRQRST v-Rel or p59^(v-rel) thetransforming protein, product of the v-rel (SEQ ID NO: 41) oncogene ofthe avian reticuloendotheliosis retrovirus strain T (Rev-T). v-Rel NLSadded to the normally cytoplasmic β-galactosidase directed that proteinto the nucleus. PFLDRLRRDQK NS1 protein of influenza A virus, thataccumulates in nuclei of virus- (SEQ ID NO: 42) infected cells.Determined to be an NLS by deletion mutagenesis of PKQKRKMAR NS1 inrecombinant SV40. The 1st NLS is conserved among all NS1 (SEQ ID NO: 43)proteins of influenza A viruses. SVTKKRKLE (SEQ ID NO: 44) Human laminA. Dimerization of lamin A was proposed to give a complex with two NLSsthat was transported more efficiently. SASKRRRLE Xenopus lamin A. NLSinferred from its similarity to human lamin A (SEQ ID NO: 45) NLS.TKGKRKRID Xenopus lamin L_(I). NLS inferred from its sequence similarityto (SEQ ID NO: 46) human lamin A NLS. CVRTTKGKRKRIDV Xenopus laminL_(I). This synthetic peptide crosslinked to chicken (SEQ ID NO: 47)bovine albumin and microinjected into HeLa cells caused nuclearlocalization. ACIDKRVKLD Human c-myc oncoprotein. This synthetic peptidecrosslinked to (SEQ ID NO: 48) chicken bovine albumin and microinjectedinto HeLa cells caused nuclear localization. ACIDKRVKLD Human c-myconcoprotein. Conjugation of the M1 peptide to human (SEQ ID NO: 49)serum albumin and microinjection of Vero cells gives complete (M1, fullypotent NLS) nuclear accumulation. M2 gave slower and only partialnuclear RQRRNELKRSP localization. (SEQ ID NO: 50) (M2, medium potencyNLS) SALIKKKKKMAP Murine c-abl (IV) gene product. The p160^(gag/v-abl)has a cytoplasmic (SEQ ID NO: 51) and plasma membrane localization,whereas the mouse type IV c-abl protein is largely nuclear. PPKKRMRRRIEAdenovirus 5 DBP (DNA-binding protein) found in nuclei of infected (SEQID NO: 52) cells and involved in virus replication and early and lategene PKKKKKRP (SEQ ID NO: 53) expression. Both NLS are needed, anddisruption of either site impaired nuclear localization of the 529 aminoacid protein. YRKCLQAGMNLEARKTKK Rat GR, glucocorticoid receptor (795amino acid) NLS1 determined by KIKGIQQATA (497-524 amino fusion withβ-galactosidase (116 kD). NLS1 is 100% conserved acid) between human,mouse and rat GR. Whereas the 407-615 amino acid (SEQ ID NO: 54)fragment of GR specifies nuclear location, the 407-740 amino acidfragment was cytoplasmic in the absence of hormone, indicating thatsequence 615-740 may inhibit the nuclear location activity. A second(NLS2) is localized in an extensive 256 amino acid C-terminal domain.NLS 2 requires hormone binding for activity. RKDRRGGRMLKHKRQRDD Human ER(estrogen receptor, 595 amino acid) NLS. NLS is betweenGEGRGEVGSAGDMRAMIN the hormone-binding and DNA-binding regions; ER, incontrast with O ACIDNLWPSPLMIKRSKK GR, lacks a second NLS. Can direct afusion product with β- (amino acid 256-303) galactosidase to thenucleus. (SEQ ID NO: 55) RKFKKFNK Rabbit PG (progesterone receptor).100% homology in humans; F→L (SEQ ID NO: 56) change in chickens. Whenthis sequence was deleted, the receptor became cytoplasmic but could beshifted into the nucleus by addition of hormone; in this case thehormone mediated the dimerization of a mutant PG with a wild type PGmolecule. GKRKNKPK (SEQ ID NO: 57) Chicken Ets1 core NLS. Within a 77amino acid C-terminal segment 90% homologous to Ets2. When deleted bydeletion mutagenesis at the gene level the mutant Ets1 becamecytoplasmic. PLLKKIKQ (SEQ ID NO: 58) c-myb gene product; directspuruvate kinase to the nucleus. PPQKKIKS (SEQ ID NO: 59) N-myc geneproduct; directs puruvate kinase to the nucleus. PQPKKKP (SEQ ID NO: 60)p53; directs puruvate kinase to the nucleus. SKRVAKRKL c-erb-A geneproduct; directs puruvate kinase to the nucleus. (SEQ ID NO: 61)CGGLSSKRPRP Adenovirus type2/5 E1a. This synthetic peptide conjugatedwith a (SEQ ID NO: 62) bifunctional crosslinker to chicken serum albumin(CSA) and microinjected into HeLa cells directed CSA to the nucleus.MTGSKTRKHRGSGA Yeast ribosomal protein L29. Double-strandedoligonucleotides (SEQ ID NO: 63) encoding the 7 amino acid peptides(underlined) and inserted at the N- MTGSKHRKHPGSGA terminus of theβ-galactosidase gene resulted in nuclear import. (SEQ ID NO: 64) RHRKHP(SEQ ID NO: 65) Mutated peptides derived from yeast L29 ribosomalprotein NLS, KRRKHP (SEQ ID NO: 66) found to be efficient NLS. The lasttwo are less effective NLS, KYRKHP (SEQ ID NO: 67) resulting in bothnuclear and cytoplasmic location of β-galactosidase KHRRHP (SEQ ID NO:68) fusion protein. KHKKHP (SEQ ID NO: 69) RHLKHP (SEQ ID NO: 70) KHRKYP(SEQ ID NO: 71) KHRQHP (SEQ ID NO: 72) PETTVVRRRGRSPRRRTPSP Double NLSof hepatitis B virus core antigen. The two underlined RRRRSPRRRRSQS (SEQID arginine clusters represent distinct and independent NLS. MutagenesisNO: 73) showed that the antigen fails to accumulate in the nucleus onlywhen (One sequence, C-terminus) both NLS are simultaneously deleted ormutated. ASKSRKRKL Viral Jun, a transcription factor of the AP-1complex. Accumulates in (SEQ ID NO: 74) nuclei most rapidly during G2and slowly during G1 and S. The cell cycle dependence of viral but notof cellular Jun is due to a C→S mutation in NLS of viral Jun. This NLSconjugated to rabbit IgG can mediate cell cycle-dependent translocation.GGLCSARLHRHALLAT Human T-cell leukemia virus Tax trans-activatorprotein. The most (SEQ ID NO: 75) basic region within the 48 N-terminalsegment. Missense mutations in this domain result in its cytoplasmicretention. DTREKKKFLKRRLLRLDE Mouse nuclear Mx1 protein (72 kD), Inducedby interferons (among (604-620) 20 other proteins). Selectively inhibitsinfluenza virus mRNA (SEQ ID NO: 76) synthesis in the nucleus and virusmultiplication. The cytoplasmic Mx2 has R→S and R→E changes in thisregion. CGYGPKKKRKV (SV40 large Synthetic peptides crosslinked to bovineserum albumin (BSA) and T) (SEQ ID NO: 77) introduced into MCF 7 or HeLaS3 cells with viral co-internalization CGYGDRNKKKKE (human method usingadenovirus serotype 3B induced nuclear import of BSA. retinoic acidreceptor) (SEQ ID NO: 78) CGYGARKTKKKIK (human glucocorticoid receptor)(SEQ ID NO: 79) CGYGIRKDRRGGR (human estrogen receptor) (SEQ ID NO: 80)CGYGARKLKKLGN (human androgen receptor) (SEQ ID NO: 81) RKRQRALMLRQARHuman XPAC (xeroderma pigmentosum group A complementing 30-42 protein)involved in DNA excision repair. By site-directed (SEQ ID NO: 82)mutagenesis and immunofluorescence. NLS is encoded by exon 1 which isnot essential for DNA repair function. EYLSRKGKLEL (SEQ ID T-DNA -linkedVirD2 endonuclease of the Agrobacterium NO: 83) tumefacienstumor-inducing (T_(i)) plasmid. A fusion protein with β- (at theN-terminus) galactosidase is targeted to the nucleus. The T-plasmidintegrates into plant nuclear DNA; VirD2 produces a site-specific nickfor T integration. VirD2 also contains a bipartite NLS at its C-terminus(see Table 2). KKSKKKRC (SEQ ID NO: 84) Putative core NLS of yeast TRM1(63 kD) that encodes the tRNA (95-102) modification enzymeN²,N²-dimethylguanosine-specific tRNA methyltransferase. Localizes atthe nuclear periphery. The 70-213 amino acid segment of TRM1 causesnuclear localization of β- galactosidase fusion protein in yeast cells.Site-directed mutagenesis of the 95-102 peptide resulted in itscytoplasmic retention. TRM1 is both nuclear and mitochondrial. The 1-48amino acid segment specifies mitochondrial import. PQSRKKLR (SEQ ID NO:85) Max protein; specifically interacts with c-Myc protein. Fusion of126-151 segment of Max to chicken pyruvate kinase (PK) gene, includingthis putative NLS, followed by transfection of COS-1 cells and indirectimmunofluorescence with anti-PK showed nuclear targeting. QPQRYGGGRGRRW(SEQ ID Gag protein of human foamy retrovirus; a mutant that completelylacks NO: 86) this box exhibits very little nuclear localization; bindsDNA and RNA in vitro.

TABLE 4 “Bipartite” or “split” NLS Signal Oligopeptide Protein andfeatures C-terminus Xenopus nucleoplasmin. Deletion analysisdemonstrated the presence of a signal responsible for nuclear location.TKKAGQAKKK (SEQ ID NO: 87) Xenopus nucleoplasmin TKKAGQAKKKKLD Xenopusnucleoplasmin. Whereas these 17 amino acids had NLS (SEQ ID NO: 88)activity, shorter versions of the 17 amino acid sequences were unable tolocate pyruvate kinase to the nucleus. TKKAGQAKKK(KLD) Xenopusnucleoplasmin. This 14 amino acid segment was (SEQ ID NO: 89) identifiedas a minimal nuclear location sequence but was unable to locate puruvatekinase to the nucleus; three more amino acids at either end (shown inparenthesis) were needed. CGQAKKKKLD Xenopus nucleoplasmin-derivedsynthetic peptide; crosslinked to (SEQ ID NO: 90) chicken serum albuminand microinjected to HeLa cells specified nuclear localization. Thissuggests that nucleoplasmin may possess a simple NLS. KRPAMINO ACIDXenopus nucleoplasmin bipartite NLS. Two clusters of basicTKKAGQAKKKK (SEQ ID NO: 91) amino acids (underlined) separated by 10amino acid are half NLS components. HRKYEAPRHx₆PRKR (SEQ ID Yeast L3ribosomal protein (387 amino acid) N-terminal 21 NO: 92) amino acid.Possible bipartite NLS. (Ribosomal proteins are transported to thenucleus to assemble with nascent rRNA). Fusion genes withβ-galactosidase were used to transform yeast cells followed byfluorescence staining with b-gal antibody. The 373 amino acid of L3fused to β-gal failed to localize to the nucleus, unless a 8 amino acidbridge containing a proline was inserted between L3 and β-gal.NKKKRKLSRGSSQKTKGTSASAK SV40 Vp3 structural protein. (35 amino acidC-terminus). By ARHKRRNRSSRS (one sequence) DEAE-dextran-mediatedtransfection of TC7 cells with mutated (SEQ ID NO: 93) constructs.RVTIRTVRVRRPPKGKHRK Simian sarcoma virus v-sis gene product (p28^(sis)).The cellular (SEQ ID NO: 94) counterpart c-sis gene encodes a precursorof the PDGF B-chain (platelet-derived growth factor). The NLS is 100%conserved between v-sis gene product and PDGF. This protein is normallytransported across the ER; introduction of a charged amino acid withinthe hydrophobic signal peptide results in a mutant protein that istranslocated into the nucleus. Puruvate kinase-NLS fusion product istransported less efficiently than cytoplasmic v-sis mutant proteins tothe nucleus. KRKIEEPEPEPKKAK Putative bipartite NLS of Xenopus laevisprotein factor xnf7. (SEQ ID NO: 95) Inferred by similarity to thebipartite NLS of nucleoplasmin. During oocyte maturation xnf7 iscytoplasmic until mid-blastula- gastrula stage due to highphosphorylation. Partial dephosphorylation results in nuclearaccumulation. KKYENVVIKRSPRKRGRPRKD Yeast SWI5 gene product, atranscription factor. Underlined (SEQ ID NO: 96) basic amino acid showsimilarity to bipartite NLS of Xenopus nucleoplasmin. The SWI5 gene istranscribed during S, G2 and M phases, during which the SWI5 proteinremains cytoplasmic due to phosphorylation by CDC28-dependent histone H1kinase at three serine residues two near and one (double underlined) inthe NLS. Translocated at the end of anaphase/G1 due to dephosphorylationof NLS. NLS confers cell cycle-regulated nuclear import ofSWI5-β-galactosidase fusion protein. MKRKRNS 735-741 Bipartite NLS ofinfluenza virus polymerase basic protein 2 (SEQ ID NO: 97) (PB2).Mutational analysis of PB2 and transfection of BHK cellsGIESIDNVMGMIGILPDMTPSTEM showed that both regions are involved innuclear import. SMRGVRISKMGVDETSSAEKIV Deletion of 449-495 region givesperinuclear localization to the 449-495 (SEQ ID NO: 98) cytoplasmicside. AHRARRLH (SEQ ID NO: 99) “Tripartite” or “doubly bipartite” NLS ofadenovirus DNA 6-13 (BSI) polymerase (AdPol). BSI and II functionedinterdependently as PPRRRVRQQPP (SEQ ID NO: 100) an NLS for the nucleartargeting of AdPol, for which BSIII was 23-33 (BSII) dispensable.BSII-III was more efficient NLS than BSI-II. PARARRRRAP (SEQ ID NO: 101)39-48 (BSIII) KRKx₁₁KKKSKK 207-226 Human poly(ADP-ribose) polymerase(116 kD). The linear (SEQ ID NO: 102) distance between the two basicclusters is not crucial for NLS activity in this bipartite NLS. Lysine222 (double underlined) is an essential NLS component. DNA binding andpoly(ADP- ribosyl)ating active site are independent of NLS.GRKRAFHGDDPFGEGPPDKKGD Herpes simplex virus ICP8 protein (infected-cellprotein). This (SEQ ID NO: 103) C-terminal portion of ICP8 introducedinto pyruvate kinase (PK) caused nuclear targeting in transfected Verocells. Inclusion of additional ICP8 regions to PK led to inhibition ofnuclear localization. KRPREDDDGEPSERKRARDDR Bipartite NLS of VirD2endonuclease of rhizogenes strains of (SEQ ID NO: 104) Agrobacteriumtumefaciens. Within the C-terminal 34 amino acid. Each region(underlined) independently directs β- glucuronidase to the nucleus, butboth motifs are necessary for maximum efficiency. VirD2 is tightly boundto the 5′ end of the single stranded DNA transfer intermediate T-strandtransferred from Agrobacterium to the plant cell genome.

TABLE 5 “Nonpositive NLS” lacking clusters of arginines/lysines Signaloligopeptide Protein and features QLVWMACNSAMINO Influenza virusnucleoprotein (NP). The underlined region ACIDFEDLRVLSFIRGTKVS (327-345)when fused to chimpanzee a₁-globin at the cDNA level and PRG 327-356microinjected into Xenopus oocytes specifies nuclear localization. (SEQID NO: 105) MNKIPIKDLLNPQ Yeast MAT a2 repressor protein, containing ahomeodomain. (NLS1 at N-terminus) (SEQ ID The two NLS are distinct, eachcapable of targeting β-galactosidase to NO: 106) the nucleus. However,deletion of NLS2 results in a2 accumulation at VRILESWFAKNIEN the pores.NLS1 and 2 may act at different steps in a localization PYLDT (NLS2 atamino acid pathway. Part of the homeodomain mediates nuclearlocalization in 141-159, part of the addition to DNA binding. The corepentapeptide containing proline and homeodomain) two other hydrophobicamino acids flanked by lysines or arginines (SEQ ID NO: 107)(underlined) was suggested as one type of NLS core. Rx₇Kx₁₅KIPRx₃HFYDrosophila HP1 (206 amino acids) that binds to EERLSWYSDNED (SEQ IDheterochromatin and is involved in gene silencing. NLS identified by β-NO: 108) galactosidase/HP1 fusion proteins introduced by P-elementmediated 152-206 (C-terminal transformation into Drosophila embryos.segment) FVx₇₋₂₀ Adenovirus type 5 E1A internal,developmentally-regulated MxSLxYMx₄MF NLS. This NLS functions in Xenopusoocytes but not in somatic cells. This NLS can be utilized up to theearly neurula stage.

TABLE 6 Nucleolar localization signals (NoLS) Signal oligopeptideProtein and features

Nucleolus localization signal in amino terminus of human p27^(X−III )proteinalso called Rex) of T cell leukemia virus type I (HTLV-I). When this peptide is fused to N-terminus of β-galactosidase, directs it to the nucleolus. Deletion of residues 2-8 (underlined), 12-18 (double-underline) or substitution of the central RR (dotted-underlined) with TT abolish nucleolar localization. Other amino acids between positions 20-80 increase nucleolar localization efficiency.RLPVRRRRRRVP (SEQ ID NO: 110)Adenovirus pTP1 and pTP2 (preterminal proteins, 80 kD)between amino acid residues 362-373. The 140 kD DNApolymerase of adenovirus when it has lost its own NLS canenter the nucleus via its interaction with pTP. The staining wasnuclear and nucleolar with some perinuclear staining as well.The NLS fused to the N-terminus of E. coli β-galactosidase wasfunctional in nuclear targeting. GRKKRRQRRRPHIV (human immunodeficiency virus) Tat protein; localizes(SEQ ID NO: 111)pyruvate kinase to the nucleolus. Tat is constitutively nucleolar.RKKRRQRRR(AHQ) Tat positive trans-activator protein of HIV-1 (humanNucleolar localization signalimmunodeficiency virus type 1). The 3 amino acids shown in(SEQ ID NO: 112)parenthesis are essential for the localization of the β-galactosidase to the nucleolus. The 9 amino acid basic region isable to localize β-gal to the nucleus but not to the nucleolus.KRVKLDQRRRP (SEQ ID NO: 113)Artificial sequence from c-Myc and HIV Tat NLSs thateffectively localizes pyruvate kinase to the nucleolus.FKRKHKKDISQNKRAVRRHuman HSP70 (heat shock protein of 70 kD); localizes pyruvate(SEQ ID NO: 114)kinase to the nucleus and nucleolus. HSP70 is physiologicallycytoplasmic but with heat-shock HSP70 redistributes to thenucleoli, suggesting that the nucleolar targeting sequence iscryptic at physiological temperature and is revealed under heat- shock.RQARRNRRRRWRERQR (35-50)HIV-1 Rev protein (116 amino acid, nucleolar). Mutations in(SEQ ID NO: 115)either of the two regions of arginine clusters severely impairnuclear localization. β-galactosidase fused to R₄W was targetedto the nucleus, and fused to the entire 35-50 region, was targetedto the nucleolus. RQARRNRRRRWRERQRQ (35-51)HIV-1 Rev protein. A fusion of this Rev peptide with β- (SEQ ID NO: 116)galactosidase became nuclear but not nucleolar. The 1-59 aminoacid segment of Rev fused to β-galactosidase localized entirelywithin the nucleolus. Whereas the NRRRRW (bold) isresponsible for nuclear targeting, the RR and WRERQRQ(double underlined) specify nucleolar localization. Rev mayfunction to export HIV structural mRNAs from the nucleus tothe cytoplasm.

TABLE 7 Karyophilic clusters on non-membrane protein kinasesNon-membrane Karyophilic peptides protein kinase Species Features 73FVVHKRCHE Protein kinase C (673 Bovine, human Known to translocate tothe (SEQ ID NO: 117) aa) β type nucleus following treatment of 96DDPRSKHKFKIH cells with mitogens. (SEQ ID NO: 118) 577 TKHPGKRLG (SEQ IDNO: 119) 71 FVVHRRCHEF Protein kinase C (697 bovine, human γ (SEQ ID NO:120) aa) type 95 DDPRNKHKFRLH (SEQ ID NO: 121) 591 TKHPAKRLG (SEQ ID NO:122) 72 FVVHKRCHE Protein kinase C (673 rabbit type α and β (SEQ ID NO:123) aa) 96 DDPRSKHKFKIH (SEQ ID NO: 124) 577 TKHPGKRLG (SEQ ID NO: 125)71 FVVHRRCHE PKC-I (701 aa) rat brain (SEQ ID NO: 126) 95 DDPRNKHKFRLH(SEQ ID NO: 127) 594 TKHPGKRLG (SEQ ID NO: 128) 22 GENKMKSRLRKG Proteinkinase C Drosophila 14 exons, 20 kb; 3 transcripts in (not conserved)(639aa, 75 kDa) adult flies; not expressed in 0-3 h (SEQ ID NO: 129)Drosophila embryos; the 80SYVVHKRCHEYVT VVHKRCHE (SEQ ID (conserved) NO:133)motif (or VVHRRCHE (SEQ ID NO: 130) (SEQ ID NO: 134)) is conserved211PDDKDQSKKKTR among all PKC known. TIK (not conserved) (SEQ ID NO:131) 614PPFKPKIKHRKMC P (not conserved) (SEQ ID NO: 132) 148 KKVLQDKRFKGlycogen synthase rat brain Phosphorylates glycogen synthase,NRELQIMRKLD (SEQ kinase 3 c-Jun, c-Myb; two isoforms ID NO: 135) GSK-3αencoded by discrete genes; highly (483 aa) expressed in brain; bothα and β GSK-3β forms are cytosolic but also (420 aa) associated with theplasma membrane consistent with their role in signal transduction fromthe cell surface. LQDRRFKNRELQ Zw3 Drosophila Product of the segmentpolarity (SEQ ID NO: 136) zeste-white 3 gene zw3; the protein encodedhas 34% homology to cdc2; mutations in zw3 give embryos that lack mostof the ventral denticles, differentiated structures derived from themost anterior region of each segment. 289ECLKKFNARRKL Ca²⁺/calmodulin-rat brain Composed of nine 50 kDa α- KGAIL dependent protein subunitsand three 60 kDa β- (SEQ ID NO: 137) kinase II (CaM kinase subunits;both are catalytic; II) β subunit (542aa, calmodulin- and ATP-binding60.3 kDa) domains; highly expressed in forebrain neurons, concentratedin postsynaptic densities; acts as a Ca²⁺-triggered switch and could beinvolved in long-lasting changes in synapses. 290LKKFNARRKL CaM kinaseII (478 rat brain This particular isoform is KGAILTTM (SEQ ID aa, 54kDa) exclusively expressed in the brain; NO: 138) α-subunit high enzymelevels in specific 450EETRVWHRRDGK brain areas; might be involved in(SEQ ID NO: 139) short- and long-term responses to transient stimuli.185 GFAKRVKGRT CADPK catalytic bovine (cardiac By Edman degradation ofprotein WTLCG subunit (349 aa, 40.6 kDa) muscle) fragments; mediates theaction of (SEQ ID NO: 140) and is activated by cAMP; consists of tworegulatory (R) and two catalytic (C) subunits; cAMP releases the Csubunit from the inactive R₂C₂ cADPK; two cDNAs were cloned encoding twoisoforms of the catalytic subunit of cADPK in mouse. 186 GFAKRVKGRTWCADPK bovine cDNA was isolated by screening a TLCG (catalytic subunit)bovine pituitary cDNA library; (SEQ ID NO: 141) (350 aa) 93% sequencesimilarity to known bovine cADPK; represents the second gene for thecatalytic subunit of cADPK. 29 EEEIQELKRKLH CGDPK (SEQ ID bovine lung Byprotein sequencing; composed KCQSVLP (SEQ ID NO: 144) of two identicalsubunits activated NO: 142) (670 aa, 76.3 kDa) in an allosteric mannerby binding 389 KILKKRHIVDTR of cGMP and not by dissociation (SEQ ID NO:143) of catalytic subunit as in cADPK; sequence similar to cADPK 117KTLKKHTIVK TPK3 S. cerevisiae cAMP-DPK is a tetrameric protein (SEQ IDNO: 145) (398 aa) with two catalytic and two cADPK regulatory subunits;cAMP activates the kinase by dissociating the catalytic subunits fromthe tetramer; all three TPK 1, 2, 3 are catalytic subunits. 16S₂H₁₃GHG₂SNF1 (633aa, 72 kDa) S. cerevisiae Ser/Thr kinase; 166 EYCHRHKIVHRDautophosphorylated; plays a LKP (SEQ ID NO: 146) central role is carboncatabolite 495 PLVTKKSKTRWH repression in yeast required for FG (SEQ IDNO: 147) expression of glucose-repressible genes; region 60-250 showshigh sequence similarity to cAMP- dependent protein kinase (cADPK). 70PVKKKKIKREIK Casein kinase II (α- Drosophila CKII is composed of α and β(SEQ ID NO: 148) subunit, catalytic) melanogaster subunits in a α₂β₂130-150 kDa 269 DILQRHSRKRW (336aa) Drosophila protein; the α-subunit isthe ERF (SEQ ID NO: 149) CKII (β-subunit, melanogaster catalytic and theβ is 146 PKSSRHHHTDG regulatory) (215aa) autophosphorylated. (SEQ ID NO:150) 142 PKSSRHHHTDG CKII (β-subunit, bovine (lung) (SEQ ID NO: 151)regulatory) (209aa, 24.2 kDa) 108 PKQRHRKSLG KIN1 (1064 aa, 117 kDa) S.cerevisiae 30% aa similarity to bovine (SEQ ID NO: 152) cADPK and 27%(KIN1) or 25% 129 GSMCKVKLAK (KIN2) aa similarity to v-Src HRYTNE withinthe kinase domain; the (SEQ ID NO: 153) catalytic domains of KIN1 and506 DRKHAKIRNQ KIN2 are near the N-terminus and (SEQ ID NO: 154) arestructural mosaics with features 638 GNIFRKLSQRR characteristic of bothTyr and KKTIEQ Ser/Thr kinases. (SEQ ID NO: 155) 773 PPLNVAKGRKL HP (SEQID NO: 156) 87 ELRQFHRRSLG KIN2 (1152 aa, 126 kDa) S. cerevisiae (SEQ IDNO: 157) 111 GKVKLVKHRQ TKE (SEQ ID NO: 158) 217 GSLKEHHARKF ARG (SEQ IDNO: 159) 807 LSVPKGRKLHP (SEQ ID NO: 160) 60FLRRGIKKKLTLD STE7 (515 aa)S. cerevisiae Implicated in the control of the (SEQ ID NO: 161) threecell types in yeast: (a, α, 472 PSKDDKFRHWC and a/α) of which a andα cells are RKIKSKIKEDKRIKRE haploid and are specialized for (SEQ ID NO:162) mating whereas a/α cells are diploid and are specialized formeiosis and sporulation; with the exception of the mating type locus,MAT, all cells contain the same DNA sequences. STE7 gene producesinsensitivity to cell- division arrest induced by the yeast matinghormone, α-factor. 722 QRRVKKLPSTTL S6KIIα (733aa) Xenopus (SEQ ID NO:163) QRRVKKLPSITL S6KII α Xenopus (SEQ ID NO: 164) 742 QRRVKKLPSTTLS6KII (752 aa) Chicken (SEQ ID NO: 165) 713QRRVRKLPSTTL S6KII (724aa)Mouse (SEQ ID NO: 166) 16GVVYKGRHKTTG CDC2Hs Human Isolated byexpressing a human (SEQ ID NO: 167) (297aa) cDNA library in S. pombe and120 FCHSRRVLHRD p34^(cdc2) selecting for clones that LKP (SEQ ID NO:168) complement a mutation in the cdc2 yeast gene; the human CDC2 genecan complement both the inviability of a null allele of S. cerevisiaeCDC28 and cdc2 mutants of S. pombe; CDC2 mRNA appears after that ofCDK2. GVVYKARHKLSGR cdc2 (297aa) S. pombe High homology to S. cerevisiae(SEQ ID NO: 169) CDC28. 119HSHRVLHRDLKP CDK2 (cell division Human Thehuman CDK2 protein has 65% (SEQ ID NO: 170) kinase 2) (298 aa) sequenceidentity to human p34^(cdc2) and 89% sequence identity to Xenopus Eg1kinase; human CDK2 was able to complement the inviability of a nullallele of S. cerevisiae CDC28 but not cdc2 mutants in S. pombe. CDK2mRNA appears in late G1/early S. 109 FCHSHRVLHRD Eg1 (297aa) XenopusCdk2-related LKP (SEQ ID NO: 171) 125 GIAYCHSHRILH CDC28 (298a) S.cerevisiae The homolog of S. pombe Cdc2 RDLKP (SEQ ID NO: 172) 119HSHRVIHRDLKP cdk3 (305aa) Human (SEQ ID NO: 173) 56 KELKHKNIVR PSSALRE(291 aa) Human cdc2-related kinase. (SEQ ID NO: 174) (SEQ ID NO: 175) 1MDRMKKIKRQ (N- PCTAIRE-1 (496 aa) Human cdc2-related kinase. terminus)(SEQ ID NO: 176) 141 DKPLSRRLRRV (SEQ ID NO: 177) 1 MKKFKRR PCTAIRE-2(523 aa) Human cdc2 related kinase. (SEQ ID NO: 178) 129 RNRIHRRIS (SEQID NO: 179) 172 SRRSRRAS (SEQ ID NO: 180) 304 HRRKVLHR (SEQ ID NO: 181)512 GHGKNRRQSM LF (SEQ ID NO: 182) 163 HTRKILHR PCTAIRE-3 Human cdc2related kinase. (SEQ ID NO: 183) (380 aa) 369 PGRGKNRRQSIF (SEQ ID NO:184) 69 EVFRRKRRLH KKIALRE (358 aa) Human cdc2-related kinase. (SEQ IDNO: 185) (SEQ ID NO: 187) 302 DKPTRKTLRKSR KHH (SEQ ID NO: 186) 1MVKRHKNT nim1⁺ gene product S. pombe (SEQ ID NO: 188) (new inducer of 87DGELFHYIRKHGP mitosis); protein (SEQ ID NO: 189) kinase (370 aa) 114DAVAHCHRFRFR HRD (SEQ ID NO: 190) 295 KKSSSKKVVRRL QQRDD (SEQ ID NO:191) 194 PAQKLRKKNNFD Wee1⁺ gene product S. pombe The Wee1⁺ genefunctions as a (SEQ ID NO: 192) (877aa) dose-dependent inhibitor that388 KQHRPRKNTNFT delays the initiation of mitosis PLPP (SEQ ID NO: 193)until the yeast cell has attained a 592 KYAVKKLKVKF certain size; Wee1has a protein SGP (SEQ ID NO: 194) kinase consensus probably regulatingcdc2 kinase. 266 PNETRRIKRAN CDC7 (497 aa) S. cerevisiae Required formitotic but not RAG (SEQ ID NO: 195) meiotic DNA replication presumablyto phosphorylate specific replication protein factors; implicated in DNArepair and meiotic recombination; some homology with CDC28 and oncogeneprotein kinases but differs in a large region within the phosphorylationreceptor domain. 48YDHVRKTRVAIKK ERK1 (MAP kinase) Rat Known totranslocate to the (SEQ ID NO: 196) (367 aa; 42 kDa) nucleus followingtheir activation by phosphorylation at T-190, and Y-192 (T-183, Y-185 inERK2). 59 ILKHFKHE FUS3 (353aa) S. cerevisiae MAP-(ERK1)-related. (SEQID NO: 197) 252 QIKSKRAKEY KSS1 (368 aa) S. cerevisiaeMAP-(ERK1)-related. (SEQ ID NO: 198) ELVKHLVKHGSN SWI6 S. cerevisiaeActivator of CACGA-box with (SEQ ID NO: 199) (803aa, 90 kDa) sequencesimilarity to cdc10; GKAKKIRSQLL required at START of cell cycle. (SEQID NO: 200) EQRLKRHRIDVSDED cdc10 S. pombe (SEQ ID NO: 201) SNIKSKCRRVV(SEQ ID NO: 202) 37 PPKRIRTD CTD kinase (528 aa) S. cerevisiae Consistsof 3 subunits of 58, 38, (suggested by the 58 kDa subunit and 32 kDa;disruption of the 58 kDa authors) (SEQ ID (catalytic) gene gives cellsthat lack CTD NO: 203) kinase, grow slowly, are cold 492 KLARKQKRPsensitive, but have different (SEQ ID NO: 204) phosphorylated forms ofRNA pol II. 29 GVSSVVRRCIHKP Phosphorylase kinase Rabbit (skeletal (SEQID NO: 205) (catalytic subunit) muscle) (386aa) 489 KKYMARRKW Myosinlight chain Chicken gizzard Ca²⁺/calmodulin-activated; QKTGHAV kinase(MLCK) (669 phosphorylated by cADPK; first (SEQ ID NO: 206) aa)described as responsible for the phosphorylation of a specific class ofmyosin light chains; required for initiation of contraction in smoothmuscle. 314 PWLNNLAEKAK Myosin light chain Rabbit (skeletal By proteinsequencing. RCNRRLKSQ kinase (partial 368 muscle) (SEQ ID NO: 207)carboxy-terminal aa 334 ILLKKYLMKRR sequence) WKKNFIAVS (SEQ ID NO: 208)28 GVSSVVRRCIHKP Phosphorylase kinase Mouse (muscle) Glycogenolyticregulatory enzyme; (SEQ ID NO: 209) (PhK) (catalytic γ undergoes complexregulation; subunit) (389 aa) composed of 16 subunits containingequimolar ratios of α, β, γ and δ subunits; high levels in skeletalmuscle; isoforms in cardiac muscle and liver; cDNA probe does nothybridize to X chromosome in mice and is thus distinct from the mutantrecessive PhK deficiency that results in glycogen storage disease.

TABLE 8 Nuclear localization signals on DNA repair proteins GeneEquivalent protein Putative NLS product in other species Features HIGHEREUKARYOTES None ERCC1 RAD10 297aa; DBD; interacts (N-terminus) stronglywith ERCC4 (XPF) MDPGKDKEGvpqpsgppaRKKF to form an excision (bipartiteNLS) endonuclease; unless the (SEQ ID NO: 210) KDKx₁₁RKK is a bipartiteNLS it may depend upon its binding with ERCC4 for its nuclear import.None ERCC2 RAD3 (S. cer) 760 aa; DNA helicase 681DKRFARGDKRGKLPR (XPD)component of TFIIH, (near the C-terminus) (four essential for cellviability; positive, one negative over a contains one nucleotide-heptapeptide stretch) binding, one DNA-binding, (SEQ ID NO: 211) andseven domains characteristic of helicases; 52% identity with S. cer RAD3at the amino acid level. 8 DRDKKKSRKRHYEDEE ERCC3 SSL2 (S cer) 782 aa;helicase, component (SEQ ID NO: 212) (XPB) Haywire (Dros) of TFIIHessential for cell 522 YVAIKTKKRILLYTM viability; helix-turn-helix, (SEQID NO: 213) DNA-BD, and helicase (weak NLS if at all, hydrophobicdomains environment) 769 PSKHVHPLFKRFRK (SEQ ID NO: 214) 84KKQTLVKRRQRKD ERCC5 RAD2; 1186 aa in human, 1196 in X. laevis; (SEQ IDNO: 215) (XPG) Rad13 3′ incision 210 EFTKRRRTL endonuclease; involved in(SEQ ID NO: 216) homologous recombination; 390 DESMIKDRKDRLP stronglynuclear (SEQ ID NO: 217) 1170 GKKRRKLRRARGRK RKT (SEQ ID NO: 218)253PQKQEKKPRKIMLNEASG ERCC6 RAD26 1493aa; involved in the (SEQ ID NO:219) CS-B preferential repair of active 314 PNKKARVLSKKEERLKK genes;nonessential for cell HIKKLQKR (SEQ ID NO: 220) viability 406PLPKGGKRQKKVP (SEQ ID NO: 221) 455 DGDEDYYKQRLRRWNK LRLQDKEKRLKLEDDSEESD(SEQ ID NO: 222) 1028 DVQTPKCHLKRRIQP X₈PKRKKFP (SEQ ID NO: 223) 1180KHKSKTKHHSVAEEETL EKHLRPKQKPKX₁₅PHLVKK RRY (SEQ ID NO: 224) 1324PAGKKSRFGKKRN (SEQ ID NO: 225) 21 PASVRASIERKRQRALM XPA RAD14 273 aa;zinc finger domain; LRGAR (SEQ ID NO: 226) involved in lesion 160PPLKFIVKKNPHHSQW recognition GD (weak) (SEQ ID NO: 227) 210NREKMKQKKFDKKVKE (weak because of F) (SEQ ID NO: 228) 72 YLRRAMKRFN(weak) XPC RAD4 (23% identity, 823 aas, 92.9 kDa; very (SEQ ID NO: 229)44% similarity) hydrophilic protein; might be 262 PSAKGKRNKGGRKKRSKinvolved in lesion PSSSEEDEGPG (SEQ ID recognition since XPC cells NO:230) (40% of all XP cases) can 297 QRRPHGRERR (weak) repair active partsof the (SEQ ID NO: 231) genome whereas inactive and 368 RTHRGSHRKDP(weak) the nontranscribed strand of (SEQ ID NO: 232) active genes arenot repaired 384 SSSSSSSKRGKKMCSDG (SEQ ID NO: 233) 531 ALKRHLLKYE(weak) (SEQ ID NO: 234) 594 SNRARKARLAEP (SEQ ID NO: 235) 660PNLHRVARKLD (weak) (SEQ ID NO: 236) 716 ERKEKEKKEKR (SEQ ID NO: 237) 740IRERLKRRYG (SEQ ID NO: 238) 801 GGPKKTKRERK (SEQ ID NO: 239) 20KSKAKSKARREEEEED XPC 940 aa; the first 117 aa are (SEQ ID NO: 240)lacking in the Legerski and 54 GKRKRG (SEQ ID NO: 241) Peterson, (1992)XPC 69 GPAKKKVAKVTVK sequence (see above); the (SEQ ID NO: 242)following 823aa are 103 PSDLKKAHHLKRG identical. (SEQ ID NO: 243) 82EIDRRKKRPLENDGPVKK Rep-3 Swi4 (S pom) 1137aa; mismatch repair KVKKVQQKE(SEQ ID (mouse) protein; Rep-3 is in the NO: 244) Duc-1 immediate5′ flanking region 375 KENVRDKKKG (HeLa) of DHFR gene (89 bp) but (SEQID NO: 245) transcribed from the opposite 571 FGRRKLKKWVT strand; abidirectional (SEQ ID NO: 246) promoter is used for both 710PLIKKRKDEIQG transcripts. (SEQ ID NO: 247) 1091 KELEGLINTKRKRLKYF AKLW(SEQ ID NO: 248) 422 EKHEGKHQKLL (weak) hMSH2 MSH2 (S cer) humanmismatch repair (SEQ ID NO: 249) protein; homologous to S. cerevisiaeMSH2; associated with the hereditary nonpolyposis colon cancer gene onchromosome 2p16. 397 PDIRRLTKKLNKRG MSH2 (SEQ ID NO: 250) (S cer) 547DAKELRKHKKYIE (SEQ ID NO: 251) 869 VKMAKRKANE (SEQ ID NO: 252) 95GELAKRSERRAEAE Human Rad2 Rad2 (S. pom) 400 aa; required for fidelity(SEQ ID NO: 253) of chromosome separation at 354 KRKEPEPKGSTKKKAKmitosis; limited similarity to TG (SEQ ID NO: 254) RAD2 (ssDNAnuclease), 394 GKFKRGK (SEQ ID rad13, and XPG (ERCC5). NO: 255) Nonemouse 339 aa; recombination-repair RAD51 protein; 83% homology to Scerevisiae RAD51 and 55% homology to E. coli RecA. None HHR23B/ RAD23Subunit of XPC (125 kDa) p58 None HHR23A RAD23 Subunit of XPC (125 kDa)32 PSQAEKKSRARAQ RPA (34 kDa RPA (70, 34, and 14 kDa (SEQ ID NO: 256)subunit) subunits) might stabilize the helicase-melted DNA around thelesion; antibodies against RPA 32 kDa subunit inhibit DNA replication.GAKKRKIDDA ATPase Q1 RecQ (E. coli) 649 aa; altered in XPC cells; (SEQID NO: 257) undetermined role in repair PKKPRGKM (SEQ ID NO: 258) HMG-1Calf thymus HMG 1 EHKKKHP (SEQ ID NO: 259) (259 aa); involved in theETKKKFKDP (SEQ ID NO: 260) recognition of cisplatin EKSKKKK(E/D)₄₁ (SEQID lesions NO: 261) E₃G₂KKKKKFAK (SEQ ID NO: 262) 512 RDEKKRKQLKKAKAKSSRP1 ABF (S cer) 709 aa, 81 kDa, structure- MAKDRKSRKKP (SEQ IDspecific recognition protein NO: 263) 1; involved in recognition of 619GESSKRDKSKKKKKVKV cisplatin-induced lesions; KMEKK (SEQ ID NO: 264) alsoinvolved in Ig gene 674 GENKSKKKRRRSEDSEE recombination; one HMG- EE(SEQ ID NO: 265) box, similarity to SRY, MTFII, LEF-1, TCF-1a, and ABF2.1 MPKRGKKG (SEQ ID Ref-1 Redox factor 1 from HeLa NO: 266) (HAP1) cells;37 kDa, 318 aa; apurinic/apyrimidinic (AP) endonuclease for DNA repairbut also of redox activity stimulating Jun/Fos DNA binding. 1 MPKRGKKGHAP1 ExoIII 323 aa; apurinic/apyrimidinic (SEQ ID NO: 267) (bovine) (E.coli) (AP)-endonuclease ExoA (S. pneumoniae) DROSOPHILA 1MGPPKKSRKDRSGGDKF Haywire ERCC3 (XPB) helicase with 66% identity toGKKRRGQDE SSL2 (S cer) human ERCC3; flies (SEQ ID NO: 268) expressingmarginal levels of EMSYSRKRQRFLVNQG Haywire display motor (weak) (SEQ IDNO: 269) defects and reduced life span YYEHRKKNIGSVHPLFK KFRG(bipartite) (SEQ ID NO: 270) 77 ARGKKKQPK (SEQ ID Rrp1 HAP1Recombination repair protein NO: 271) 1); 679 aa; the 252 aa C- 98KPKGRAKKA (SEQ ID terminal domain is NO: 272) homologous to AP- 157QAKGRKKKELP (SEQ ID endonucleases, whereas the NO: 273) 1-426 aa domainis highly 179 EPPKQRARKE (SEQ ID charged, carries all of the NO: 274)putative NLSs. 241 PPKAASKRAKKGK (SEQ ID NO: 275) 282 PKKRAKKTT (SEQ IDNO: 276) 317 EPAPGKKQKKSAD (SEQ ID NO: 277) 336 EEEAKPSTETKPAKGR KKAP(SEQ ID NO: 278) 372 KPARGRKKA (SEQ ID NO: 279) 394 GSKTTKKAKKAE (SEQ IDNO: 280) S. CEREVISIAE 200 IEKRRKLYISGG RAD1 ERCC4 1100 aa; 30% sequence(SEQ ID NO: 281) (XPF) identity to Rad16; RAD1 515 NKKRGVRQVLLN (SEQRad16 interacts strongly with ID NO: 282) RAD10 565 KEQVTTKRRRTRG(conserved in Rad16) (SEQ ID NO: 283) 1024 NLRKKIKSFNKLQ (SEQ ID NO:284) 89 RQRKERRQGKRE RAD2 XPGC 1031 aa, 117.8 kDa; ssDNA (SEQ ID NO:285) Rad13 endonuclease; rad mutants 907 ENKFEKDLRKKLVNNE are defectivein incision (SEQ ID NO: 286) 984 RDVNKRKKKGKQKRI (SEQ ID NO: 287) 1017KRISTATGKLKKRKM (SEQ ID NO: 288) 672 GKDDYGVMVLADRRF RAD3 ERCC2 or XPD;778 aa, 89,779 Da; 30% SRKRSQLP (contains the bulky (S. cer) Rad15 orRhp3 sequence identity to rad16; F) (SEQ ID NO: 289) ATP-dependent DNAhelicase; single-stranded DNA-dependent ATPase. 26 PLSRRRRVRRKNQPLPDRAD4 XPC 754 aa; mutations in RAD4 AKKKFKTG (SEQ ID NO: 290) that thatinactivate the 134 NEERKRRKYFHMLYL excision repair function of (SEQ IDNO: 291) RAD4 result in truncated 160 EWINSKRLSRKLSNL proteins missingthe C- (weak) (SEQ ID NO: 292) terminal one-third of RAD4. 254EMSANNKRKFKTLKRSD weak (SEQ ID NO: 293) 382 WMNSKVRKRRITKDDF GEK (SEQ IDNO: 294) 403 RKVITALHHRKRTKID DYED (SEQ ID NO: 295) 504KTGSRCKKVIKRTVGRP (SEQ ID NO: 296) 150 FHPKRRRIYGFR (SEQ ID RAD5 1169aa; helicase involved in NO: 297) postreplication-repair (RAD6 215DSRGRKKASM (SEQ ID epistasis group); binds DNA NO: 298) with the sevenhelicase 297 DGESLMKRRRTEGGNK motifs and with zinc fingers; REK (SEQ IDNO: 299) increases the instability of 1152 DEDERRKRRIEE poly (GT)repeats in the yeast (SEQ ID NO: 300) genome. 1 MSTPARRRLMRDFKRM RAD6RAD6 mediates the KEDAPP (SEQ ID NO: 301) ubiquitination of H2A and H2Bhistones 15 GVAKLRKEKSGAD RAD10 ERCC1 210 aa; forms an (SEQ ID NO: 302)endonuclease with RAD1; 76 DDYNRKRPFRSTRPGK the basic and tyrosine-rich(SEQ ID NO: 303) central domain was suggested to bind DNA by ionicinteractions and tyrosine intercalation. 172 EGKAHRREKKYE RAD14 XPAC247aa, 29.3 kDa; two zinc (SEQ ID NO: 304) fingers; involved in lesion200 NRLREKKHGKAHIHH recognition; 27% sequence (SEQ ID NO: 305) identityand 54% sequence similarity (if conserved residues are grouped together)to human XPA; deletion of RAD14 gene generates high UV sensitivity. 345ERRKQLKKQGPKRP Ixr1 591 aa; two consecutive (SEQ ID NO: 306) (S. cer)HMG boxes; involved in 479 ETYKKRIKEWESCYPDE recognition of1,2-intrastrand (SEQ ID NO: 307) d(GpG) and d(ApG) cisplatin crosslinks.None RAD23 HHR23 483 LTCKKLKTHNRIILSG RAD26 ERCC6 1075aa; disruption ofthe weak (SEQ ID NO: 308) (yeast CS-B (hum) RAD26 gene gives viable 934NALRKSRKKITKQYEIGT ERCC6) yeast cells unable to PX₉GEIRKRDPpreferentially repair the (SEQ ID NO: 309) actively transcribed strands;surprisingly, in contrast to human CS-B cells, disruption of the RAD26in yeast does not cause sensitivity to UV, Cisplatin, or X-rays. 634KPTSKPKRVRTATKKKIP MRE11 Rad32 (S pom) meiotic recombination (SEQ ID NO:310) protein; functions in the 408 FYKKRSPVTRSKKSG same pathway withRAD51 (SEQ ID NO: 311) none; RAD51 RecA (E. coli) 402 aa; essential forrepair of 361 GFKKGKGCQR DSBs and recombination; (SEQ ID NO: 312)associates strongly with RAD52; self associates; neither RAD51 nor RAD52possess a typical simple NLS. none; RAD51 364 aa 328 GFKKGKGCQR (K.lactis) (SEQ ID NO: 313) none; RAD52 Rad22 504 aa; rad52 mutants are 155ERAKKSAVTDALKRSLR defective in ionizing GFGX₈DKDFLAKIDKVKFDP radiation,mitotic PD (tripartite) recombination, mating-type (SEQ ID NO: 314)switching, and repair of DSDs. 1 MARRRLPDRPP RAD54 898 aa;recombination-repair (SEQ ID NO: 315) protein; ATP-binding motif; 65GGRSLRKRSA helicase domains; in the (SEQ ID NO: 316) same subfamily ofhelicases 99 QLTKRRKD with MOT1 and SNF2. (SEQ ID NO: 317) 269DETVFVKSKRVKASSS RAD55 Similarity to RecA, and (extremely weak if at allNLS) lower similarity to RAD51, (SEQ ID NO: 318) RAD57, and DMC1 317GEDRKREGRNLKR (SEQ ID NO: 319) 371 PISRQSKKRKFDYRVP RAD57 460 aa;nucleotide-binding (SEQ ID NO: 320) domain; limited similarity to RAD5162 GLKKPRKKTKSSRH SSL2 ERCC3 (XPB) 843 aa; putative helicase that (SEQID NO: 321) seems to function in repair 688 GRILRAKRRNDEG but also inthe removal of (SEQ ID NO: 322) secondary structures in the 5′ 784GRGSNGHKRFKS (weak) untranslated region of mRNA (SEQ ID NO: 323) toallow ribosome binding and scanning. 50 TRRHLCKIKGLSE (weak) DMC1 RecA334 aa; yeast homolog of (SEQ ID NO: 324) RecA, meiosis-specific; 277DGRKPIGGHX₁₂RKGRG dmc1 mutants are defective DER (bipartite) (SEQ ID inreciprocal recombination NO: 325) and accumulate DSBs 11 ETEKRCKQKEQRYPMS1 904 aa, 103 kDa; mismatch- (SEQ ID NO: 326) repair protein; MutL(Salmonella) and HexB (Streptococcus) homolog None HRR25 Hhp1, Hhp1 (Spom) Mutations in HRR25 Ser/Thr 1 MDLRVGRKFRIGRKIG CKI (mamm proteinkinase cause defects (SEQ ID NO: 327) in DNA repair and 139GRRGX₈GLSKKYRDFNT retardation in cell cycling HRHIP (Bipartite weak NLS)(SEQ ID NO: 328) 96 HELTKRSSRRVETEK YKL510 383 aa; structure-specific(SEQ ID NO: 329) endonuclease; two domains of about 100 aa with sequencesimilarity to N- and C-terminal regions of RAD2. 200 MLAMARRKKKMSAK MOT1Modifier of transcription 1; (SEQ ID NO: 330) 1867 aa; DNA helicase ofS. cerevisiae 617 EHYKVKHTEK (weak required for NLS) (SEQ ID NO: 331)viability; increases gene 670 LHPEKKRSISE (weak expression of several.,but NLS) (SEQ ID NO: 332) not all, pheromone- responsive genes in theabsence of STE12; the 1257 to 1825 aa domain (568 aa residues) hashomology to SNF2 and RAD54 S. POMBE 60 SSIDEx₅SIKRKRRI (SEQ ID Swi4Duc-1 113 kDa; KCII sites are NO: 333) Rep-3 upstream of NLS like inSV40 large T; the homologous prokaryotic MutS and HexA lack NLS 96GELAKRVARHQKARE Rad2 380 aa (weak NLS) (SEQ ID NO: 334) 362 GSAKRKRDS(SEQ ID NO: 335) 372 KGGESKKKR (SEQ ID NO: 336) None Rad9 — 427 aa; nohomology to other DNA repair proteins; rad9 fission yeast mutants aresensitive to both UV and ionizing radiation; may be involved inrecombination- repair. None Rhp3 or ERCC2 772 aa; DNA helicase; 65% 681DKRYGRSDKRTKLPK rad15 RAD3 identity to RAD3 and 55% (SEQ ID NO: 337)identity to ERCC2; essential for viability 464 PPSKRRRVRGG Rad 16 RAD1Function in repair of UV (SEQ ID NO: 338) damage for both cyclobutanedimer and (6-4) photoproduct lesions; Rad16 interacts with Swi10. 431DFKQAILRKRKNESPE Rad21 628 aa, 67.8 kDa, acidic EVEP (SEQ ID NO: 339)protein; a single base substitution in mutant rad21-45, changing an Ileinto a Thr, is responsible for the low efficiency in repair of DSBsafter g-radiation although capable of arresting at G2. 490 DKKAKKG (SEQID Rad22 RAD52 496 aa; functions in NO: 340) recombination-repair andmating-type switching. 394 DVVQFYLKKKYTRSKRN Rad32 MRE11 (S cer) 648 aa;meiotic DG (weak because of Y) (SEQ recombination protein; rad32 ID NO:341) mutants are sensitive to g- 575 PSPALLKKTNKRRELP and UV radiation;functions (SEQ ID NO: 342) in the same pathway with Rhp51 (RAD51). Rad51recombination-repair GLAKKYRDHKTHLHIP (weak Hhp1 CKI (mamm) Ser/Thrprotein kinase; NLS because of Y and H) (SEQ HRR25 (S cer) mutation inthis gene causes ID NO: 343) repair defects None Hhp2 CKI (mamm) Ser/Thrprotein kinase; GLAKKYRD 

 KTHVHIP (H in HRR25 (S cer) mutation in this gene causes Hhp1 isreplaced by F in Hhp2) repair defects (SEQ ID NO: 344)

TABLE 9 NLS in Transcription factors NLS and FlanksProtein factor and features highly basic HR₄QRTRK₇R (SEQ ID NO: 345)Human GCF (GC-factor) LRRKSRP (SEQ ID NO: 346) SRRTKRRQ (SEQ ID NO: 347)GRKRKKRT (SEQ ID NO: 348)Oct-6 protein transcription factor from mouse cellsGRRRKKRT (SEQ ID NO: 349)Mouse Oct-2 protein transcription factors (Oct-2.1 for Oct-2.6 isoforms)ARKRKRT (SEQ ID NO: 350) Oct-3 from mouse P19 embryonal carcinoma cellsNRRQKGKRS (SEQ ID NO: 351) ECRRKKKE (SEQ ID NO: 352)Human ATF-1. In basic region/leucine zipper. ERKKRRRE (SEQ ID NO: 353)Human ATF-3 (in basic region that binds DNA)AKCRNKKKEKT (SEQ ID NO: 354) SKKKIRL (SEQ ID NO: 355)Mouse Pu.1 (Friend erythroleukemia cells). Related to ets oncogeneQKGNRKKM (SEQ ID NO: 356) VKKVKKKL (SEQ ID NO: 357)VKRKKI (SEQ ID NO: 358) Human PRDII-BF1 that binds to IFN-βgene promoter. (The largest CRNRYRKLE (SEQ ID NO: 359)DNA-binding protein known, of 298 kD). IRKRRKMK (SEQ ID NO: 360)PKKKRLRL (SEQ ID NO: 361) GKKKKRKREKLMurine LEF-1 (397 aa). Lymphoid-specific with an HMG1-like box.(within the HMG-box) NLS is identical to that of human TCF- 1α.(SEQ ID NO: 362) GKKKKRKREKL Human TCF-lα (399 aa) (within the HMG-box)(T cell-specific transcription factor that activates the T cell (SEQ ID NO: 363)receptor Cα). Contains an HMG box. NLS core is identical to that of murine LEF-1. GKKKRRSREKH Human TCF-1 (within the HMG-box) (SEQ ID (uniquely T cell-specific). HMG box containing. NO: 364)PKKCRARF (SEQ ID NO: 365) FKQRRIKL (SEQ ID NO: 366)Xenopus laevis Oct-1(within POU-domain) NRRRKKRT (SEQ ID NO: 367)NRRQKEKRI (SEQ ID NO: 368) DKRSRKRKRSK (SEQ ID NO: 369)Drosophila Suvar (3) 7 gene product involved in position-effectRLRIDRKRN (SEQ ID NO: 370)variegation (932 aas). Five widely spaced zinc-fingers could helpAKRSRRS (SEQ ID NO: 371) condensation of the chromatin fiber.IRKRRKMKSVGD₂E₂ (SEQ ID NO:372)Human MBP-1 (class I MHC enhancer binding protein 1) mw 200(not suggested as NLS by thekD. Induced by phorbol esters and mitogens in Jurkat T cells.authors; between the 1st and  2nd zinc finger) PPKKKRLRLAE(suggested as NLS by the authors; just before 2nd zincfinger) (SEQ ID NO: 373) CRNRYRKLE (within 1st zinc finger)(SEQ ID NO: 374) PRRKRRV (SEQ ID NO: 375)rat TTF-1 (thyroid nuclear factor that binds to the promoter ofHRYKMKRQ (SEQ ID NO: 376)thyroid-specific genes). An homeodomain protein.DGKRKRKN (SEQ ID NO: 377) Human thyroid hormone receptor α(c-erbA-1 gene). Belongs to the DDSKRVAKRKL (SEQ ID NO: 378)family of cytoplasmic proteins that are receptors of hydrophobicNRERRRKEE (SEQ ID NO: 379) ligands such as steroids, vitD, retinoic acid, thyroid hormones. WKQRRKF (SEQ ID NO: 380)The ligand binding may expose the NLS for nuclear import of thereceptor-ligand complex. NRRKRKRS (SEQ ID NO: 381)Drosophila gcl (germ cell-less) gene product (569 aa, 65 kD), PKKKKL (SEQ ID NO: 382)located in nuclei, required for germ line formation.

C. elegans Sdc-3 protein (sex-determining protein) (2,150 aas). A zinc finger protein. LKKIRRKIKNKI (SEQ ID NO: 392)Drosophila BBF-2 (related to CREB/ATF) ESRRKKKE (SEQ ID NO: 393)Group 0000 DRNKKKKE (SEQ ID NO: 394)Xenopus RAR (retinoic acid receptor) ARRRRP (SEQ ID NO: 395)GRRRRA (SEQ ID NO: 396)Human ATF-2 (the 2nd and 3rd NLS are in basic region that bindsDEKRRKV (SEQ ID NO: 397) DNA) CRQKRKV (SEQ ID NO: 398)ERKRRD (SEQ ID NO: 399)Myn (murine homolog of Max). Forms a specific DNA-bindingSRKKLRME (SEQ ID NO: 400)complex with c-Myc oncoprotein through a helix-loop-helix/leucinezipper. EEKRKRTYE (SEQ ID NO: 401)  human NFκB p65 (550 aa).Not binding DNA; complexed with p50 that binds DNA. NFκB p50also contains a NLS (Table 3b). GRRRRA (SEQ ID NO: 402)Human HB16, a cAMP response element-binding proteinDEKRRKF (SEQ ID NO: 403) SRCRQKRKV (SEQ ID NO: 404)SKKKKTKV (SEQ ID NO: 405) Human TFIIE-β(general transcription initiation protein factor; NRPDKKKI (SEQ ID NO: 406) forms tetramer α₂β₂ with TFIIE-α)QRRKKP (SEQ ID NO: 407) QKKRRFKT (SEQ ID NO: 408)SRKRKM (SEQ ID NO: 409)Human kup transcriptional activator (433 aas). Two distantly spaced zinc fingers. Expressed in hematopoietic cells and  testis.ERKRLRNRLA (SEQ ID NO: 410)Mouse Jun-B homologue to avian sarcoma virus 17 oncogene v-junATKCRKRKL (SEQ ID NO: 411)product. One region is similar to yeast GCN4 and to Fos. (19 aa stretch)DKRx₆ERKRRD (N-terminus)Max (specifically associates with c-Myc, N-Myc, L-Myc). The Max-(SEQ ID NO: 412)Myc complex binds to DNA; neither Max nor Myc alone exhibitQSRKKLRME (C-terminus) appreciable DNA binding. (SEQ ID NO: 413)

Chicken VBP (vitellogenin gene-binding protein). Leucine zipper.Related to rat DBP.

Xenopus borealis B1 factor. Closely related to the mammalian USF.Binds to CACGTG in TFIIIA promoter to developmentally regulateits expression.

Human USF (upstream stimulatory factor) activating the major lateadenovirus promoter YRRYPRRRG (SEQ ID NO: 421)YB-1, a protein that binds to the MHC class II Y box. YB-1 QRRPYRRRRF (SEQ ID NO: 422) is a negative regulator.YRPRFRRG (SEQ ID NO: 423) QRRYRRN (SEQ ID NO: 424)YRRRRP (SEQ ID NO: 425)

Human TFEB Binds to IgH enhancer.

Human TFE3 (536 aa). Binds to μE3 enhancer of IgH genes.KTVALKRRKASSRL (SEQ ID  Human Dr1 (176 aa, 19 kD). Interacts with TBP (TATA-binding NO: 431)protein) thus inhibiting association of TFIIA and/or TFIIB with TBP. TBP-Dr1 association is affected by Dr1 phosphorylation to repress activated and basal transcription. 1 LRRRGRQTY (SEQ ID NO: 432)Drosophila ultrabithorax protein (from the conserved 61 amino acid27 LTRRRRIEM (SEQ ID NO: 433)homeodomain segment only). Conserved in the antenappedia51 QNRRMKLKKEI (SEQ ID   homeodomain protein. NO: 434)SNRRRPDHR (SEQ ID NO: 435) C. elegans sex-determining Tra-1 protein. Zinc finger. Peaks in VYRGRRRVRRE (SEQ ID  the second larval stage. NO: 436)P₇AP₂RRRRSADNKD₂ (SEQ  ID NO: 437) PKKPRHQF (SEQ ID NO: 438)EKRKKERN (SEQ ID NO: 439)Yeast NPS1 transcription protein factor (1359 aa) involved in LLRRLKKEVE (SEQ ID cell growth control at G2 phase. Has a catalytic domain of  NO: 440)protein kinases. EPLGRIRQKKRVY₂D₂ (SEQ  ID NO: 441)(EDAIKKRREARERRRLRQ)  (SEQ ID NO: 442) DKETTASRSKRRSSRKKRT(SEQ ID NO: 443) ESKKKKPKL (SEQ ID NO: 444) KKTAAKKTKTKS (SEQ ID NO: 445) QRKRQKL (SEQ ID NO: 446)Human 243 transcriptional activator (968 aas), induced by mitogensKAKKQK (SEQ ID NO: 447)in T cells. N-terminal half is homologous to oncoprotein Rel andLRRKRQK (SEQ ID NO: 448)Drosophila Dorsal protein involved in development. The C-terminalhalf contains repeats found in proteins involved in cell-cycle control of yeast and tissue differentiation in Drosophila.RDIRRRGKNKV (SEQ ID NO: 449)Mouse NF-E2 (45 kD), an erythroid transcription factor from mouseQNCRKRKLE (SEQ ID NO: 450)erythroleukemia (MEL) cells. Involved in globin gene regulation.Binds to AP-1-like sites. Homology to Jun B, GCN4, Fos, ATF1 andCREB in basic region/leucine zipper (see FIG. 2). Group 000x00DKIRRKN (SEQ ID NO: 451) Human glucocorticoid receptorARKTKKKI (SEQ ID NO: 452) 473 DKIRRKNCP (SEQ ID Mouse and human GR (glucocorticoid recptor) NO: 453)EARKTKKKIKGIQ (SEQ ID  NO: 454) Group 000x0

C/EBP (CCAAT/enhancer binding protein).Functions in liver-specific gene expression. DKIRRKN (SEQ ID NO: 458)Human mineralocorticoid receptor ARKSKKL (SEQ ID NO: 459)DKIRRKN (SEQ ID NO: 460) Human PR (progesterone receptor)GRKFKKF (SEQ ID NO: 461) EEVQRKRQKLMP (SEQ ID Human and mouse NFκB 105 kD precursor of p50 (968 aas) (first R NO: 462)is at 361 position). EEVQRKRQKL (SEQ ID Human NF-κB p50 (DNA-binding subunit). Identical to protein NO: 463)KBF1, homologous to rel oncogene product. NF-κB p65 alsocontains a NLS (Table 3a). GKTRTRKQ (SEQ ID NO: 464)Human TEF-1 (SV40 transcriptional enhancer factor 1). 426 aa.ARRKSRD (SEQ ID NO: 465)

Rat, mouse, human IRF-1 (interferon regulatory factor-1). Induced in lymphoma T cells by the pituitary peptide hormone prolactin.Regulates the growth-inhibitory interferon genes.GKCKKKN (SEQ ID NO: 468)Ehrlich ascites S-II transcription factor. A general factor that acts a tthe elongation step.

Tobacco TAF-1 transcriptional activator YKLDHMRRRIETDE (SEQ ID Drosophila TFIIEα (433 aa), a general transcription factor for  NO: 472)RNA polymerase II. Composed of subunits α and β.

Human ER (estrogen receptor); 595 aa. EQRRHRIE (SEQ ID NO: 476)Yeast ADA2 (434 aa), a potential transcriptional adaptor required TTRAEKKRLL (SEQ ID for the function of certain acidic activation domains. NO: 477)IDKKRSKEAKE (SEQ ID  NO: 478) EAALRRKIRTISKYeast GCN5 gene product (439 aa), required for the function of(SEQ ID NO: 479)GCN4 transcriptional activator and for the activity of the HAP2-3-4 complex. Group 00x00 NKKMRRNRF (SEQ ID NO: 480) Mouse LFB3NRRKx₄RQK (SEQ ID NO: 481) TKKGRRNRF (SEQ ID NO: 482) Mouse LFB1NRRKx₄RHK (SEQ ID NO: 483) NKKMRRNRFK (SEQ ID  rat vHNF1-A NO: 484)NKKMRRNR (SEQ ID NO: 485) murine HNF-1β TKKGRRNRF (SEQ ID NO: 486)mouse HNF-1 NKKMRRNRF (SEQ ID NO: 487) human vHNF1TKKGRRNRF (SEQ ID NO: 488) rat liver HNF1 LRRQKRFK (SEQ ID NO: 489)rat HNF-3β QQH₃SH₄Q (SEQ ID NO: 490) LRRQKRFK (SEQ ID NO: 491)rat HNF-3γ LRRQKRFK (SEQ ID NO: 492) rat HNF-3α

rat DBP a protein factor that binds to the D site of the albumin gene promoter

rat AT-BP1. Highly acidic domain. Two zinc fingers. Binds to theB-domain of α_(l)-antitrypsin gene promoter and to the NF-κB site inthe MHC gene enhancer. DRRVRKGKV (SEQ ID NO: 496)A 19 kD Drosophila melanogaster nonhistone associated withheterochromatin. SKHGRRARRLDP (SEQ ID murine EBF (early B-cell factor) of 591 aa. Regulates the pre-B NO: 497)and B lymphocyte-specific mb-1 gene. Expressed in pre-B and B-celllines but not in plasmocytomas, T-cell and nonlymphoid cell lines.GRRTRRE (SEQ ID NO: 498) human Sp1

yeast SNF2, a transcriptional regulator of many genes. Group 0x00x0

mouse AGP/EBP (87% similarity to C/EBP), ubiquitously expressed

rat LAP, a 32-kD liver-enriched transcriptional activator, also present in lung, with 71% sequence similarity to C/EBP. Leucine zipper. Accumulates to maximal levels around birth.

Ig/EBP-1 (immunoglobulin gene enhancer-binding protein). Formsheterodimers with C/EBP.

mouse c-Myb DYYKVKRPKTD (SEQ ID NO: 512)Drosophila eyes absent protein (760 aa), a nuclear protein thatGRARGRRHQ (SEQ ID NO: 513)functions in early development to prevent programmed cell death FRYRKIKDIY (SEQ ID NO: 514)and to allow the event that generate the eye to proceed. Mutations cause programmed cell death of eye progenitor cells. Group 0x0x00AKAKAKKA (SEQ ID NO: 515) rat IL-6DBP interacting with interleukin-6 responsive elements. Has a leucine zipper domain. DKRQRNRC (SEQ ID NO: 516) mouse H-2RIIBP (MHC class I genes H-2 region II binding protein).FkrtirkD Member of the nuclear hormone receptor superfamily. FkrtirkDchicken RXR, related to RAR (retinoic acid receptor), a nuclearDKRQRNRC (SEQ ID NO: 517) protein factor from the thyroid/steroid hormone receptor family

human NF-IL6 (345 aa). Specifically binds to ILl-responsiveelement in the IL-6 gene. Leucine zipper. Homology to C/EBP.QKKNRNKC (SEQ ID NO: 521) mouse PPAR (peroxisome proliferator activated receptor) Group 000xx00EQIRKLVKKHG (SEQ ID  yeast RAP1 NO: 522)It binds regulatory sites at yeast mating type silencers.FRRSMKRKA (SEQ ID NO: 523) human vitamin D receptor (427 aa)Group 00xx00 LKRHQRRH (SEQ ID NO: 524)mouse WT1 (the murine homolog of human Wilms' tumorpredisposition gene WT1) LKRHQRRH (SEQ ID NO: 525) human WT33 (Wilms'tumor predisposition) Group 000xx0

yeast SWI3 99 kd), highly acidic protein. Global transcriptionactivator. EVLKVQKRRIYD (SEQ ID human RBAP-1 (retinoblastoma-associated protein 1) factor (412  NO: 527)aa). A protein that binds to the pocket (functional domain) of the retinoblastoma (RB) protein involved in suppression of cell growth (tumor suppressor). The transcription factor E2F, implicated in cell growth, binds to the same pocket of RB.

TABLE 10 NLS in other nuclear proteins Putative NLS ProteinYKSKKKA (SEQ ID NO: 528) Yeast L3 TKKLPRKT (SEQ ID NO: 529)TRKKGGRRGRRL (SEQ ID NO: 530) Yeast 59 ribosomal protein C-terminusARATRRKRCKG (SEQ ID NO: 531) Yeast L16 ribosomal proteinGKGKYRNRRW (SEQ ID NO: 532) yeast L2 ribosomal protein (homologous toXenopus L1). Encoded by intronless genes.

Xenopus laevis L1 ribosomal protein (homologousto yeast L2) Encoded by intronless genes. ERKRKS (SEQ ID NO: 539)human S6 ribosomal protein (homologous to yeastGKRPRTKA (SEQ ID NO: 540) S10) HKRRRI (SEQ ID NO: 541)LKKQRTKKNKE (SEQ ID NO: 542) PKMRRRTYR (SEQ ID NO: 543)Rat L17 ribosomal protein (184 aas) KKKISQKKLKK (SEQ ID NO: 544)YMRRRTYRA (SEQ ID NO: 545) Podocoryne carnea (hydrozoan, Coelenteratum)EVKKVSKKKL (SEQ ID NO: 546) L17 ribosomal protein (184 aas) highlyhomologous to rat L17.

human, rat ribosomal S13 protein ERKRKS (SEQ ID NO: 548)yeast S10 ribosomal protein (homologous to humanQRLQRKRH (SEQ ID NO: 549) S6) IRKRRA (SEQ ID NO: 550)GRRRKKHRSRSRSRERRSRSRDRGRG₁₂GRER 35 kD subunit of U2 small nuclearDRRRSRDRER (SEQ ID NO: 551)ribonucleoprotein auxiliary factor (U2AF), anessential mammalian splicing factor. U2AF³⁵interacts with the 65 kD subunit (U2AF⁶⁵). Bothproteins are concentrated in a small number ofsubnuclear organelles, the coiled bodies.

human UsnRNP-associated 70 k protein (437 aas)that is phosphorylated at Arg/Ser-rich domains; involved in splicingQKRNNKKSKKKRCAE (SEQ ID NO: 558) yeast TRM1 enzyme for the N²,N²-EKLRKLKI (near C-terminus) (SEQ ID NO: 559)dimethylguanosine modification of bothmitochondrial and cytoplasmic tRNAs. TRM1 isboth nuclear and mitochondrial. The first motif is within a region (70-213 aa segment) known tocause nuclear localization of β-galactosidase. NKRKRV (SEQ ID NO: 560)Yeast nucleoporin NUP1 (1076 aa, 113 kD); anSLKNRSNRKRE (SEQ ID NO: 561)integral component of the pore complex. InvolvedEPKRKRRLP (SEQ ID NO: 562)in both binding and translocation steps of nuclearARMRHSKR (C-terminus) (SEQ ID NO: 563) import.KAEKEx₃KVD₂E₂ (SEQ ID NO: 564) Chicken, Xenopus No 38 nucleolar (38 kD);Kx₃Kx₅Kx₃R (SEQ ID NO: 565)involved in intranuclear packaging of preribosomalparticles. Shuttles between nucleus and cytoplasm.KTEREAEKALEEKx₇R (SEQ ID NO: 566)Chicken, hamster nucleolin (92 kD). BindsKx₅Kx₇Kx₄RX3EDTTEETLR (SEQ ID NO: 567) preribosomal RNA. Shuttles between nucleus andRG₂RG₂RG₃RG₂FG₂RG₃RGFG₂RG₃FRG₂RG₄ cytoplasm.DHKPQGKKIKFE (SEQ ID NO: 568) (C-terminus) WYKHFKKTKD (SEQ ID NO: 569)human SATB1 (763 aa) which binds selectively to AT-rich MARs with mixedA, T, C on one strand excluding G. Binds to minorgroove with little contact with bases.

yeast CBF5p, a centromere-binding protein(55kDa, 483aa). The KKE repeat at its C-terminusoccurs in microtubule-binding domains; yeast cells containing only three copies of the KKE repeat of CBF5p delay at G₂/M; depletion of CBE5p arrests cells at G₁/S. TKKKSFKL (SEQ ID NO: 574)yeast CCE1, a cruciform cutting endonucleaseKSERERMLRESLKEERRRF (SEQ ID NO: 575)rat nucleoporin 155 or Nup155 (1390 aas, 155kDa), a protein of the nuclear pore complex;contains 46 consensus sites for various kinases;associated with both the nucleoplasmic and thecytoplasmic region of pores. PKKGSKKA (SEQ ID NO: 576)human H2B variant differentially expressed duringDGKKRKRSRKES (SEQ ID NO: 577) the cell cycle GAKRHRKVLRD(SEQ ID NO: 578) Calf thymus histone H4 14-24 (102 aa)PAIRRLARRG (SEQ ID NO: 579) 32-41 EHARRKT (SEQ ID NO: 580) 74-80ARRIRGERA 127-135 (SEQ ID NO: 581) Calf thymus H3 (135 aa)GSHHKAKGK 121-129 (SEQ ID NO: 582) Calf thymus H2A (129 aa)RGKSGKARTKAKSRSSR 3-19 (SEQ IDSea urchin Psammechinus miliaris H2A (123 aa) NO: 583)PKKGSKKA 10-17 (SEQ ID NO: 584) Calf thymus H2B QKKDGKKRKRSRKES22-36 (SEQ ID NO: 585)  (125 aa) GGKKRHRKRKGSY (SEQ ID NO: 586)Sea urchin Psammechinus miliaris H2B (122 aa) 22-34 PRTDKKRRRKRKES19-32 (SEQ ID NO: 587) Starfish H2B (121 aa)PAKAPKKKA 12-20 (SEQ ID NO: 588) Trout testis H1EAKKPAKKA 104-112 (SEQ ID NO: 589) (194 aa)AKKPKKV 128-134 (SEQ ID NO: 590) AKKSPKKAKKP 142-152 (SEQ ID NO: 591)PKKVKKP 183-189 (SEQ ID NO: 592) PRRKAKRA 30-37 (SEQ ID NO: 593)Sea urchin Parechinus angulosus sperm H1 (248PKKAKKT 119-125 (SEQ ID NO: 594) aa) AKAKKAKA 129-136 (SEQ ID NO: 595)AKKARKAKA 139-147 (SEQ ID NO: 596) AKKAKKPKKKA 171-181 (SEQ ID NO: 597)AKKAKKPAKK 182-191 (SEQ ID NO: 598) SPKKAKKP 192-199 (SEQ ID NO: 599)AKKSPKKKKAKRS 200-212 (SEQ ID NO: 600) PKKAKKA 213-219 (SEQ ID NO: 601)AKKAKKS 227-233 (SEQ ID NO: 602) PRKAGKRRSPKKARK 234-248 (SEQ IDNO: 603) ARRRKTA 1-7 (SEQ ID NO: 604) Annelid sperm H1aIRKFIRKA 55-61 (SEQ ID NO: 605) (119 aa) PKKKKA 83-88 (SEQ ID NO: 606)AKKPKAKKVKKP 89-100 (SEQ ID NO: 607) AKKKTNRARKPKTKKNR 104-120 (SEQ IDNO: 608) PKRKVSS 1-7 (SEQ ID NO: 609) Calf thymus HMG14EEPKRRSARLS 14-24 (SEQ ID NO: 610) (100 aa)PKRKAEGDAK 1-10 (SEQ ID NO: 611) Calf thymus HMG17PKGKKGKA 52-59 (SEQ ID NO: 612) (89aa; 9,247 D)PKKPRGKM (SEQ ID NO: 613) Calf thymus HMG 1 EHKKKHP (SEQ ID NO: 614)(259 aa) ETKKKFKDP (SEQ ID NO: 615) EKSKKKK(E/D)₄₁ (SEQ ID NO: 616) E₃G₂KKKKKFAK (SEQ ID NO: 617) EHKKKHP (SEQ ID NO: 618) Calf thymus HMG 2PKGDKKGKKKDP(SEQ ID NO: 619) (256 aa) E₄ G₃KKKKKFAK(SEQ ID NO: 620)PKRKSATKGDEPARR 1-15 (SEQ ID NO: 621) Trout testis H6 (60 aa)KPKKAAAPKKA 30-34 (SEQ ID NO: 622)

REFERENCES U.S. Patent Documents

-   U.S. Pat. No. 4,394,448 July, 1983 Szoka, Jr. et al.-   U.S. Pat. No. 4,598,051 July, 1986 Papahadjopoulos et al.-   U.S. Pat. No. 5,013,556 May, 1991 Woodle et al.

Journal Articles

-   Allen, T. M. and Chonn, A. (1987) “Large unilamellar liposomes with    low uptake into the reticuloendothelial system” FEBS Lett.    223:42-46.-   Allen, T. M. et al. (1991) “Liposomes containing synthetic lipid    derivatives of polyethylene glycol show prolonged circulation    half-lives in vivo” Biochim. Biophys. Acta 1066:29-36.-   Anderson, W. F. (1992) “Human gene therapy” Science 256:808-813.-   Aoki, K. et al. (1995) “Liposome-mediated in vivo gene transfer of    antisense K-ras construct inhibits pancreatic tumor dissemination in    the murine peritoneal cavity” Cancer Res. 55:3810-3816.-   Arcasoy, S. M. et al. (1997) “Polycations increase the efficiency of    adenovirus-mediated gene transfer to epithelial and endothelial    cells in vitro” Gene Ther. 4:32-38.-   Beauchamp, C. O. et al. (1983) “A new procedure for the synthesis of    polyethylene glycol-protein adducts; effects on function, receptor    recognition, and clearance of superoxide dismutase, lactoferrin, and    alpha 2-macroglobulin”Anal. Biochem. 131:25-33.-   Bongartz, J.-P. et al. (1994) “Improved biological activity of    antisense oligonucleotides conjugated to a fusogenic peptide” Nucl.    Acids Res. 22:4681-4688.-   Boulikas, T. (1993) “Nuclear localization signals (NLS)” Crit. Rev.    Eukar. Gene Expression 3:193-227.-   Boulikas, T. (1994) “Putative nuclear localization signals (NLS) in    protein transcription factors” J. Cell. Biochem. 55:32-58.-   Boulikas, T. (1996a) “Cancer gene therapy and immunotherapy”    Intl. J. Oncol. 9:941-954.-   Boulikas, T. (1996b) “Gene therapy to human diseases: ex vivo and in    vivo studies”Intl. J. Oncol. 9:1239-1251.-   Boulikas, T. (1996c) “Liposome DNA delivery and uptake by cells”    Oncol. Rep. 3:989-995.-   Boulikas, T. (1996d) “Nuclear import of protein kinases and    cyclins” J. Cell. Biochem. 60:61-82.-   Boulikas, T. (1997a) “Gene therapy of prostate cancer: p53, suicidal    genes, and other targets” Anticancer Res. 17:1471-1506.-   Boulikas, T. (1997b) “Nuclear import of DNA repair proteins”    Anticancer Res. 17:843-864.-   Boulikas, T. (1997c) “Nuclear localization signal peptides for the    import of plasmid DNA in gene therapy” Int. J. Oncol. 10:301-309.-   Boulikas, T. (1998a) “Status of gene therapy in 1997: Molecular    mechanisms, disease targets, and clinical applications” Gene Ther.    Mol. Biol. 1:1-172.-   Boulikas, T. (1998b) “Nucleocytoplasmic trafficking: implications    for the nuclear import of plasmid DNA during gene therapy” Gene    Ther. Mol. Biol. 1:713-740.-   Boulikas, T. and Martin, F. (1997) “Histones, protamine, and    polylysine but not poly(E:K) enhance transfection efficiency”    Int. J. Oncol. 10:317-322.-   Capaccioli, S. et al. (1993) “Cationic lipids improve antisense    oligonucleotide uptake and prevent degradation in cultured cells and    in human serum” Biochem. Biophys. Res. Comm. 197:818-825.-   Creuzenet, C. et al. (1997) “Interaction of alpha s2- and    beta-casein signal peptides with DMPC and DMPG liposomes” Peptides    18:463-472.-   Culver, K. W. (1996) in: Gene Therapy: A primer for physicians,    Second Edition. Mary Ann Liebert, Inc. Publications, NY, pp. 1-198.-   Curtain, C. et al. (1999) “The interactions of the N-terminal    fusogenic peptide of HIV-1 gp41 with neutral phospholipids” Eur.    Biophys. J. 28:427-436.-   de la Maza, A. et al. (1998) “Solubilization of phosphatidylcholine    liposomes by the amphoteric surfactant dodecyl betaine” Chem. Phys.    Lipids 94:71-79.-   Decout, A. et al. (1999) “Contribution of the hydrophobicity    gradient to the secondary structure and activity of fusogenic    peptides” Mol. Membr. Biol. 16:237-246.-   Duguid, J. G. et al. (1998) “A physicochemical approach for    predicting the effectiveness of peptide-based gene delivery systems    for use in plasmid-based gene therapy” Biophys. J. 74:2802-2814.-   Filion, M. C. and Phillips, N. C. (1997) “Toxicity and    immunomodulatory activity of liposomal vectors formulated with    cationic lipids toward immune effector cells” Biochim. Biophys. Acta    1329:345-356.-   Fresta, M. et al. (1998) “Liposomal delivery of a 30-mer antisense    oligodeoxynucleotide to inhibit proopiomelanocortin expression” J.    Pharm. Sci. 87:616-625.-   Gabizon, A. and Papahadjopoulos, D. (1988) “Liposome formulations    with prolonged circulation time in blood and enhanced uptake by    tumors” Proc. Natl. Acad. Sci. USA 85:6949-6953.-   Gabizon, A. et al. (1989) “Pharmacokinetics and tissue localization    of doxorubicin encapsulated in stable liposomes with long    circulation times” J. Natl. Cancer Inst. 81:1484-1488.-   Ghosh, J. K. and Shai, Y. (1999) “Direct Evidence that the    N-Terminal Heptad Repeat of Sendai Virus Fusion Protein Participates    in Membrane Fusion” J. Mol. Biol. 292:531-546.-   Green, M. and Loewenstein, P. M. (1988) “Autonomous functional    domains of chemically synthesized human immunodeficiency virus tat    transactivator protein” Cell 55:1179-1188.-   Gupta, D. and Kothekar, V. (1997) “500 picosecond molecular dynamics    simulation of amphiphilic polypeptide Ac(LKKL)₄ NHEt with 1,2    di-mysristoyl-sn-glycero-3-phosphorylcholine (DMPC) molecules”    Indian J. Biochem. Biophys. 34:501-511.-   Hofland, H. E. J. et al. (1996) “Formation of stable cationic    lipid/DNA complexes for gene transfer” Proc. Natl. Acad. Sci. USA    93:7305-7309.-   Jaaskelainen, I. et al. (1994) “Oligonucleotide-cationic liposome    interactions. A physicochemical study” Biochim. Biophys. Acta    1195:115-123.-   Judice, J. K. et al. (1997) “Inhibition of HIV type 1 infectivity by    constrained alpha-helical peptides: implications for the viral    fusion mechanism” Proc. Natl. Acad. Sci. USA 94:13426-13430.-   Kono, K. et al. (1993) “Fusion activity of an amphiphilic    polypeptide having acidic amino acid residues: generation of fusion    activity by alphα-helix formation and charge neutralization”    Biochim. Biophys. Acta 1164:81-90.-   Lambert, G. et al. (1998) “The C-terminal helix of human    apolipoprotein AII promotes the fusion of unilamellar liposomes and    displaces apolipoprotein AI from high-density lipoproteins” Eur. J.    Biochem. 253:328-338.-   Lambert, O. et al. (1998) “A new “gel-like” phase in dodecyl    maltoside-lipid mixtures: implications in solubilization and    reconstitution studies” Biophys. J. 74:918-930.-   Lappalainen, K. et al. (1997) “Intracellular distribution of    oligonucleotides delivered by cationic liposomes: light and electron    microscopic study” Histochem. Cytochem. 45:265-274.-   Lasic, D. (1997) in: Liposomes in Gene Delivery, CRC Press, pp.    1-295.-   Lee, S. et al. (1992) “Effect of amphipathic peptides with different    alpha-helical contents on liposome-fusion” Biochim. Biophys. Acta    1103:157-162.-   Lelkes, P. I. and Lazarovici, P. (1988) “Pardaxin induces    aggregation but not fusion of phosphatidylserine vesicles” FEBS    Lett. 230:131-136.-   Leonard, A. N. and Cohen, D. E. (1998) “Submicellar bile salts    stimulate phosphatidylcholine transfer activity of sterol carrier    protein 2” J. Lipid Res. 39:1981-1988.-   Lewis, J. G. et al. (1996) “A serum-resistant cytofectin for    cellular delivery of antisense oligodeoxynucleotides and plasmid    DNA” Proc. Natl. Acad. Sci. USA 93:3176-3181.-   Li, S, and Huang, L. (1997) “In vivo gene transfer via intravenous    administration of cationic lipid-protamine-DNA (LPD) complexes” Gene    Ther. 4:891-900.-   Lins, L. et al. (1999) “Molecular determinants of the interaction    between the C-terminal domain of Alzheimer's beta-amyloid peptide    and apolipoprotein E alpha-helices” J. Neurochem. 73:758-769.-   Litzinger, D. C. et al. (1996) “Fate of cationic liposomes and their    complex with oligonucleotide in vivo” Biochim. Biophys. Acta    1281:139-149.-   Lopez, O. et al. (1998) “Direct formation of mixed micelles in the    solubilization of phospholipid liposomes by Triton X-100” FEBS Lett.    426:314-318.-   Lusa, S. et al. (1998) “Direct observation of lipoprotein    cholesterol ester degradation in lysosomes” Biochem. J. 332:451-457.-   Macosko, J. C. et al. (1997) “The membrane topology of the fusion    peptide region of influenza hemagglutinin determined by    spin-labeling EPR” J. Mol. Biol. 267:1139-1148.-   Macreadie, I. G. et al. (1997) “Cytotoxicity resulting from addition    of HIV-1 Nef N-terminal peptides to yeast and bacterial cells”    Biochem. Biophys. Res. Commun. 232:707-711.-   Martin, F. and Boulikas, T. (1998) “The challenge of liposomes in    gene therapy” Gene Ther. Mol. Biol. 1:173-214.-   Martin, I. et al. (1999) “Membrane fusion induced by a short    fusogenic peptide is assessed by its insertion and orientation into    target bilayers” Biochemistry 38:9337-9347.-   Martin, I. and Ruysschaert, J. M. (1997) “Comparison of lipid    vesicle fusion induced by the putative fusion peptide of fertilin (a    protein active in sperm-egg fusion) and the NH₂-terminal domain of    the HIV2 gp41” FEBS Lett. 405:351-355.-   Massari, S, and Colonna, R. (1986) “Gramicidin induced aggregation    and size increase of phosphatidylcholine vesicles” Chem. Phys.    Lipids 39:203-220.-   Melino, S. et al. (1999) “Zn(2+) ions selectively induce    antimicrobial salivary peptide histatin-5 to fuse negatively charged    vesicles. Identification and characterization of a zinc-binding    motif present in the functional domain” Biochemistry 38:9626-9633.-   Midoux, P. and Monsigny, M. (1999) “Efficient gene transfer by    histidylated polylysine/pDNA complexes” Bioconjug. Chem. 10:406-411.-   Murata, M. et al. (1991) “Modification of the N-terminus of membrane    fusion-active peptides blocks the fusion activity” Biochem. Biophys.    Res. Commun. 179:1050-1055.-   Niidome, T. et al. (1997) “Membrane interaction of synthetic    peptides related to the putative fusogenic region of PH-30 alpha, a    protein in sperm-egg fusion” J. Peptide Res. 49:563-569.-   Pak, C. C. et al. (1999) “Elastase activated liposomal delivery to    nucleated cells” Biochim. Biophys. Acta 1419:111-126.-   Papahadjopoulos, D. et al. (1991) “Sterically stabilized liposomes:    Improvements in pharmacokinetics and antitumor therapeutic efficacy”    Proc. Natl. Acad. Sci. USA 88:11460-11464.-   Parente, R. A. et al. (1988) “pH-dependent fusion of    phosphatidylcholine small vesicles. Induction by a synthetic    amphipathic peptide” J. Biol. Chem. 263:4724-4730.-   Partidos, C. D. et al. (1996) “Priming of measles virus-specific CTL    responses after immunization with a CTL epitope linked to a    fusogenic peptide” Virology 215:107-110.-   Pecheur, E. I. et al. (1997) “Membrane anchorage brings about    fusogenic properties in a short synthetic peptide” Biochemistry    36:3773-3781.-   Peelman, F. et al. (1999) “Characterization of functional residues    in the interfacial recognition domain of lecithin cholesterol    acyltransferase (LCAT)” Protein Eng. 12:71-78.-   Pereira, F. B. et al. (1997) “Permeabilization and fusion of    uncharged lipid vesicles induced by the HIV-1 fusion peptide    adopting an extended conformation: dose and sequence effects”    Biophys. J. 73:1977-1986.-   Pillot, T. et al. (1999) “The nonfibrillar amyloid beta-peptide    induces apoptotic neuronal cell death: involvement of its C-terminal    fusogenic domain” Neurochem. 73:1626-1634.-   Pillot, T. et al. (1997) “Specific modulation of the fusogenic    properties of the Alzheimer beta-amyloid peptide by apolipoprotein E    isoforms” Eur. J. Biochem. 243:650-659.-   Pillot, T. et al. (1997) “The 118-135 peptide of the human prion    protein forms amyloid fibrils and induces liposome fusion” J. Mol.    Biol. 274:381-393.-   Plank, C. et al. (1996) “Activation of the complement system by    synthetic DNA complexes: a potential barrier for intravenous gene    delivery” Hum. Gene Ther. 7:1437-1446.-   Rodriguez-Crespo, I. et al. (1994) “Prediction of a putative fusion    peptide in the S protein of hepatitis B virus” J. Gen. Virol.    75:637-639.-   Rodriguez-Crespo, I. et al. (1999) “Fusogenic activity of    hepadenavirus peptides corresponding to sequences downstream of the    putative cleavage site” Virology 261:133-142.-   Ross, G. et al. (1996) “Gene therapy in the United States: a    five-year status report” Hum. Gene Ther. 7:1781-1790.-   Schroeder, F. et al. (1990) “Intermembrane cholesterol transfer:    role of sterol carrier proteins and phosphatidylserine” Lipids    25:669-674.-   Schroth-Diez, B. et al. (1998) “Fusion activity of transmembrane and    cytoplasmic domain chimeras of the influenza virus glycoprotein    hemagglutinin” J. Virol. 72:133-141.-   Schutze, W. and Muller-Goymann, C. C. (1998) “Phase transformation    of a liposomal dispersion into a micellar solution induced by    drug-loading” Pharm. Res. 15:538-543.-   Song, Y. K. et al. (1997) “Characterization of cationic    liposome-mediated gene transfer in vivo by intravenous    administration” Hum. Gene Ther. 8:1585-1594.-   Sorgi, F. L. et al. (1997) “Protamine sulfate enhances    lipid-mediated gene transfer” Gene Ther. 4:961-968.-   Suenaga, M. et al. (1989) “Basic amphipathic helical peptides induce    destabilization and fusion of acidic and neutral liposomes” Biochim.    Biophys. Acta 981:143-150.-   Takle, G. B. et al. (1997) “Delivery of oligoribonucleotides to    human hepatoma cells using cationic lipid particles conjugated to    ferric protoporphyrin IX (heme)” Antisense Nucleic Acid Drug Dev.    7:177-185.-   Templeton, N. S. et al. (1997) “Improved DNA: liposome complexes for    increased systemic delivery and gene expression” Nature Biotechnol.    15:647-652.-   Thierry, A. R. and Dritschilo, A. (1992) “Intracellular availability    of unmodified, phosphorothioated and liposomally encapsulated    oligodeoxynucleotides for antisense activity” Nucl. Acids Res.    20:5691-5698.-   Tirosh, O. et al. (1998) “Hydration of polyethylene glycol-grafted    liposomes” Biophys. J. 74:1371-1379.-   Torchilin, V. P. (1998) “Polymer-coated long-circulating    microparticulate pharmaceuticals” J. Microencapsul. 15:1-19.-   Torchilin, V. P. et al. (1992) “Targeted accumulation of    polyethylene glycol-coated immunoliposomes in infarcted rabbit    myocardium” FASEB J. 6:2716-2719.-   Tournois, H. et al. (1990) “Gramicidin A induced fusion of large    unilamellar dioleoylphosphatidylcholine vesicles and its relation to    the induction of type II nonbilayer structures” Biochemistry    29:8297-8307.-   Ulrich, A. S. et al. (1999) “Ultrastructural characterization of    peptide-induced membrane fusion and peptide self-assembly in the    lipid bilayer” Biophys. J. 77:829-841.-   Voneche, V. et al. (1992) “The 19-27 amino acid segment of gp51    adopts an amphiphilic structure and plays a key role in the fusion    events induced by bovine leukemia virus” J. Biol. Chem.    267:15193-15197.-   Wattiaux, R. et al. (1997) “Cationic lipids destabilize lysosomal    membrane in vitro” FEBS Lett. 417:199-202.-   Weissig, V. et al. (1998) “Accumulation of protein-loaded    long-circulating micelles and liposomes in subcutaneous Lewis lung    carcinoma in mice” Pharm. Res. 15:1552-1556.-   Zelphati, O. and Szoka, Jr., F. C. (1997) “Intracellular    distribution and mechanism of delivery of oligonucleotides mediated    by cationic lipids” Pharm. Res. 13:1367-1372.-   Zuidam, N. J. and Barenholz, Y. (1997) “Electrostatic parameters of    cationic liposomes commonly used for gene delivery as determined by    4-heptadecyl-7-hydroxycoumarin”Biochim. Biophys. Acta 1329:211-222.

1. A method for producing micelles with entrapped therapeutic agents,comprising: a) combining an effective amount of a negatively chargedtherapeutic agent with an effective amount of a cationic lipid in aratio where about 30% to about 90% the negatively charged atoms areneutralized by positive charges on lipid molecules to form anelectrostatic micelle complex in about 20% to about 80% ethanol; and b)combining the micelle complex of step a) with an effective amount of afusogenic-karyophilic peptide conjugates in a ratio range of about 0.0to about 0.3, thereby producing micelles with entrapped therapeuticagents.
 2. The method of claim 1, wherein the negatively chargedtherapeutic agent is a therapeutic agent selected from the groupconsisting of a polynucleotide and a negatively charged drug.
 3. Themethod of claim 2, wherein the polynucleotide is a DNA polynucleotide oran RNA polynucleotide.
 4. The method of claim 2, wherein thepolynucleotide is a DNA polynucleotide.
 5. The method of claim 4,wherein the DNA polynucleotide comprises plasmid DNA.
 6. The method ofclaim 1, further comprising combining an effective amount of an anioniclipid in step a).
 7. The method of claim 6, wherein the anionic lipid isdipalmitoyl phosphatidyl glycerol (DDPG) or a derivative thereof.
 8. Themethod of claim 4, further comprising combining an effective amount of aDNA condensing agent selected from the group consisting of spermine,spermidine, polylysine, polyarginine, polyhistidine, polyornithine andmagnesium or a divalent metal ion.
 9. The method of claim 5, wherein theplasmid DNA comprises a sequence encoding p53, HSV-tk, p21, Bax, Bad,IL-2, IL-12, GM-CSF, angiostatin, endostatin and oncostatin.
 10. Themethod of claim 1, wherein the cationic lipids are selected from thegroup consisting of3β-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol,dimethyldioctadecyl ammonium bromide (DDAB),N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) ammoniumbromide (DMRIE), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP),dioctadecylamidoglycylspermine (DOGS),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP),1,2-disteroyl-3-trimethylammonium propane (DSTAP).
 11. The method ofclaim 10, wherein the cationic lipids are combined with the fusogeniclipid DOPE in a molar ratio from about 1:1 to about 2:1.
 12. The methodof claim 11, wherein the cationic lipids are combined with the fusogeniclipid DOPE in a molar ratio of 1:1.
 13. The method of claim 1, whereinthe fusogenic-karyophilic peptide is an NLS peptide.
 14. The method ofclaim 13, wherein the NLS peptide is a peptide selected from the groupconsisting of Seq. ID Nos. 20-622.
 15. The method of claim 1, whereinthe fusogenic-karyophilic peptide conjugate is a sole fusogenic peptide.16. The method of claim 1, wherein the NLS peptide component of thefusogenic-karyophilic peptide conjugate is an NLS peptide selected fromthe group consisting of Seq. ID Nos. 20-622.
 17. The method of claim 1,wherein the fusogenic/NLS peptide conjugates comprise amino acidsequences selected from the group consisting of (KAWLKAF)₃ (SEQ IDNO:1), GLFKAAAKLLKSLWKLLLKA (SEQ ID NO:2), LLLKAFAKLLKSLWKLLLKA (SEQ IDNO:3) as well as all derivatives of the prototype(Hydrophobic₃Karyophilic₁Hydrophobic₂Karyophilic₁)₂₋₃ where Hydrophobicis any of the A, I, L, V, P, G, W, F and Karyophilic is any of the K, R,or H, containing a positively-charged residue every 3rd or 4th aminoacid, that form alpha helices and direct a net positive charge to thesame direction of the helix.
 18. The method of claim 1, wherein thefusogenic/NLS peptide conjugate comprise an amino acid sequence selectedfrom the group consisting of GLFKAIAGFIKNGWKGMIDGGGYC (SEQ ID NO:4) frominfluenza virus hemagglutinin HA-2 and YGRKKRRQRRR (SEQ ID NO:5) fromTAT of HIV.
 19. The method of claim 1, wherein the fusogenic/NLS peptideconjugate comprise an amino acid sequence selected from the groupconsisting of MSGTFGGILAGLIGLL(K/R/H)₁₋₆ (SEQ ID NO:6), derived from theN-terminal region of the S protein of duck hepatitis B virus but withthe addition of one to six positively-charged lysine, arginine orhistidine residues, and combinations of these, GAAIGLAWIPYFGPAA (SEQ IDNO:7) derived from the fusogenic peptide of the Ebola virustransmembrane protein; residues 53-70 (C-terminal helix) ofapolipoprotein (apo) AII peptide, the 23-residue fusogenic N-terminalpeptide of HIV-1 transmembrane glycoprotein gp41, the 29-42-residuefragment from Alzheimer's beta-amyloid peptide, the fusion peptide andN-terminal heptad repeat of Sendai virus, the 56-68 helical segment oflecithin cholesterol acyltransferase.
 20. The method of any of claim 13to 19, wherein the NLS peptide component in fusogenic/NLS peptideconjugates are synthetic peptides containing the above said NLS butfurther modified by additional K, R, H residues at the central part ofthe peptide or with P or G at the N- or C-terminus.
 21. The method ofclaim 13, wherein the fusogenic peptide/NLS peptide conjugates arelinked to each other with a short amino acid stretch representing anendogenous protease cleavage site.
 22. The method of claim 1, whereinthe structure of the preferred prototype fusogenic/NLS peptide conjugateused in this invention is: PKKRRGPSP(L/A/I)₁₂₋₂₀ (SEQ ID NO:8) where(L/A/I)₁₂₋₂₀ is a stretch of 12-20 hydrophobic amino acids containing A,L, I, Y, W, F and other hydrophobic amino acids.
 23. The method of claim1, wherein the fusogenic/NLS peptide conjugates are added to the mixtureof DNA/cationic lipid and are incorporated into micelles.
 24. The methodof claim 1, further comprising combining an effective amount of anencapsulating lipid solution to step b).
 25. The method of claim 24,wherein the encapsulating lipid is a lipid comprising cholesterol (40%),dioleoylphosphatidylethanolamine (DOPE) (20%),palmitoyloleoylphosphatidylcholine (POPC) (12%), hydrogenated soyphosphatidylcholine (HSPC) (10%), distearoylphosphatidylethanolamine(DSPE) (10%), sphingomyelin (SM) (5%), and derivatized vesicle-forminglipid M-PEG-DSPE (3%).
 26. The method of claim 24, wherein theencapsulating lipid is a liposome.
 27. The method of claim 26, whereinthe liposomes comprises vesicle-forming lipids and between about 1 toabout 7 mole percent of distearoylphosphatidyl ethanolamine (DSPE)derivatized with an effective amount of polyethyleneglycol.
 28. Themethod of claim 27, wherein the liposomes have a selected average sizeof about 80 to about 160 nm.
 29. The method of claim 27, wherein thepolyethyleneglycol has a molecular weight from about 1,000 to about5,000 daltons.
 30. A micelle with an entrapped therapeutic agentproduced by the method of claim
 1. 31. A liposome encapsulatedtherapeutic agent produced by the method of claim
 24. 32. The method ofclaim 31, wherein the therapeutic agent further comprises regulation bya liver, spleen or bone marrow regulatory DNA sequence.
 33. The methodof claim 32, wherein the regulatory DNA sequence is nuclear matrix DNAisolated from liver, spleen or bone marrow cells.
 34. A method fordelivering a therapeutic agent in vivo, comprising administration of aneffective amount of the micelle of claim 30 to a subject.
 35. The methodof claim 34, wherein the therapeutic agent further comprises regulationby a tumor-specific regulatory DNA sequence.
 36. The method of claim 35,wherein the tumor-specific regulatory sequence is nuclear matrix DNAisolated from specific tumor cells.
 37. A method for delivering atherapeutic agent in vivo, comprising administration of an effectiveamount of the liposome encapsulated agent of claim 31 to the subject.38. The method of claim 34 or 37, wherein the administration isintravenous administration or by injection.
 39. A micelle with anentrapped DNA polynucleotide produced by the method of claim
 9. 40. Amethod for reducing tumor size in a subject comprising administration ofan effective amount of the micelle of claim 39 to the subject.
 41. Themethod of claim 40, further comprising administration of an effectiveamount of a second therapeutic agent, wherein the agent is selected fromthe group consisting of ganciclovir, 5-fluorocytosine, an antisenseoligonucleotides a ribozyme, and a triplex-forming oligonucleotidedirected against genes that control the cell cycle or signalingpathways.
 42. The method of claim 41, further comprising administrationof an effective amount of a second therapeutic agent, wherein the secondtherapeutic agent is selected from the group consisting of adriamycin,angiostatin, azathioprine, bleomycin, busulfane, camptothecin,carboplatin, carmustine, chlorambucile, chlormethamine,chloroquinoxaline sulfonamide, cisplatin, cyclophosphamide, cycloplatam,cytarabine, dacarbazine, dactinomycin, daunorubicin, didox, doxorubicin,endostatin, enloplatin, estramustine, etoposide, extramustinephosphat,flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate,hydroxyurea, idoxuridine, interferons, interleukins, leuprolide,lobaplatin, lomustine, mannomustine, mechlorethamine,mechlorethaminoxide, melphalan, mercaptopurine, methotrexate,mithramycin, mitobronitole, mitomycin, mycophenolic acid, nocodazole,oncostatin, oxaliplatin, paclitaxel, pentamustine, platinum-triaminecomplex, plicamycin, prednisolone, prednisone, procarbazine, proteinkinase C inhibitors, puromycine, semustine, signal transductioninhibitors, spiroplatin, streptozotocine, stromelysin inhibitors, taxol,tegafur, telomerase inhibitors, teniposide, thalidomide, thiamiprine,thioguanine, thiotepa, tiamiprine, tretamine, triaziquone, trifosfamide,tyrosine kinase inhibitors, uramustine, vidarabine, vinblastine, vincaalcaloids, vincristine, vindesine, vorozole, zeniplatin, zeniplatin, andzinostatin.